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User Manual - Hitachi Solutions America

1. ee ji il ia TERT 7 E pie 7a a a Ma N 27 T dee A A me IA B Ic Ic2 ID HA1 142 IIB IC ID 4 Adjusted MFI of 47 4 E2 i 10 ee ee B ee oe ee i 8B 80 H Pe ped eae OI AI H ia OD A l e ASS ee 4 b Io A dun sew Nessa fe diet TSI a oe ee eee to o 20 eae i i 0 A ICM COLL E AE MGI LOOP wis Max 100 Figure 8 9 Multi Compare graphs y axis maximum 340 top y axis maximum 100 bottom 8 12 MasterPlex GT www miraibio com CHAPTER 8 GRAPHS Adjusting the Graph Width or Height 1 To change the graph width click the Hw1 0 button and select a fac tor from the drop down list The graph bars and graph width are increased or decreased by the selected factor If necessary use the scrollbar at the bottom of the Project Window to view the Multi Compare graphs 2 To change the graph height click the x1 0 button and select a factor from the drop down list The default graph bars and graph height are increased by the selected factor If necessary use the scrollbar at the right of the Project Window to view the Multi Compare graphs NOTE If the default graph view does not display the entire graph legend increase the graph height to view the complete legend Repositioning the Allele Name Tags 1 To rotate the position of the allele name tags move the slider at the top of the Project Window Figure 8 10 The
2. G Oo B O KA o 4 5 50 I 1 I 1 i E pise t renin a a a a A TA a I 734 I4 16126C 1C116294T 8 1C2163194 IIB146C IIB152C IIC 199C Adjusted MFI of h16401 8 C8 T o D Q 1 o m ORTE AE E AO Rey RERA n Re ee eres Mena a ee e 2 504 I i 2 imr amg t a Se e 734 14 16126C IC1 162941 C2163194 JIB146C IIB152C IIC 199C Figure 7 24 Multi Compare bar graphs four samples Sample ede i WL W734 14161294 1C116294T IC216319A IIB146C IIB152C IID 263A Figure 7 25 Depth bar graph for the four samples in Figure 7 15 7 26 MasterPlex GT www miraibio com CHAPTER 7 RESULTS TABLES Sample by Sample Scatter Graph The Sample by Sample scatter graph plots the background adjusted MFI for two user selected samples Each point in the graph represents an allele For more information see Sample by Sample Scatter Graph on page 8 19 1 Right click a sample you want to plot in the scatter graph and select Sort by Expression from the shortcut menu This places the selected sample in the left column of the Typing table and sorts the Typing table by expression level 2 Right click the second sample for the scatter plot and select Open Scatter Plot from the shortcut menu The Sample scatter graph is displayed Figure 7 26 R2 0 89951886 1400 k b ee ee k b 1 300 1 200 ee ae eee oo enn 1 100 1 000 Wenn aban
3. JSamoleEmotv 2 2 68 45 877 1855 co lea S22 _ Sarmnle Enty 1 To o w a o a 5 63 7 lsu 592 Samole Emoty 1 2 a 38 51 if 3 e Jaa aosa Sarmole Eat e Jaa o SameEmiw 0 2 l 0 1 a 0 1 2 3 2 2 a 3 faved 6462 Jsampetmevi 2 2 2 0 1 a 2 A4 o ea 6426 Jsampeemov l 2 2 2 0 1 2e woo w3 daaa SamoeEmy l l 2 i 3 3 2 2 4 pa fares Jos JSampietmiv 2 2 A 3 ip 2 HI 48 3 6188 Sample Empty 2 2 1 i 2 1 4 3 3 Figure 4 2 Project Window Typing table view FI MasterPlex GT Typing Sample2 Sample2 gtp File Edit View Function Option Window Help pelag ee fal slalel Ble sa 3 a fe El 2u e ele Typing Table sui Gath TTIE a EN IE ees Fy Sampled eats hoa fiere ioraa _ andersor 16217 _ 16 ple Z PET 07S CEU JN DCE J Typing Table ems Sane Ee ic il Multi Graph B lma ea Seem gt O SampleBase q Typing Table rs iaetad lems jsampetmov al Multi Graph TSI ea as Samoe Emoty aea set 5amvie Emety ae ce a a e ae Ean e e2 las Jsomoletmev e Jaa eus Samoe Emotv a besim oa a MFI Adlusted MFI Count Bead ave sb cv h esa jan Sete Emoty Sos 102 a fes fes Semple Emoty Ti I1 2 see 2 a3 ess Semple Emoty e l b jea e052 _____ Samole Emoty pace li wa jus 0 Jsampetmiv 1415 ley fara feos Jsamoietmotv az ea hor Samole Emotv 162 11 0 A2 48 4 7017 Sample Empty e CO 38 124 EC Samale Emo
4. 3 8 MasterPlex GT www miraibio com CHAPTER 3 BEFORE YOU BEGIN If necessary merge results that you want to combine and analyze in one Typing table You can merge samples and compare results across experiments sample merge or combine results from differ ent bead sets that probe the same sample layer merge Set negative controls Confirm the defaults or choose new allele calling parameters Figure 3 9 View the genotyping results in the table or graph format see Table 3 3 Save the results to a project gtp that includes analysis parameter settings graphs dendrogram and user selected samples MasterPlex GT www miraibio com 3 9 CHAPTER 3 BEFORE YOU BEGIN Table 3 3 MasterPlex GT table and graph formats Format Typing table Figure 3 10 Heat Map Figure 3 12 Allele Call window Figure 3 13 Homology table Figure 3 14 Homology chart Figure 3 15 Multi Compare bar graph Figure 3 16 Depth Bar graph Figure 3 16 Sample scatter graph Figure 3 18 Allele scatter graph Figure 3 19 Dendrogram Figure 3 20 3 10 MasterPlex GT Displays Background adjusted median fluorescence intensity MFI relative intensity RI bead count or allele frequency data A color coded map bead MFI data for each sample Four tables allele calls allele frequency genotype frequency and haplotype frequency The homology score between sample genotypes A plot of the correlation coefficients
5. 48 1 Sample Empty i 141 Folge sz Sample Emnty 48 14 e305 Jsample Empty 130 112 287 a1 beadsonynew 2253 JsampleEmetv B4 beadsnew 17787 Sample Empty beadsold 351 Samvle Emoty 2 B2 aza as Sample Emnty 153 65 135 3041 BL 4740000000 15519 Sample Empty 4 5 4 58 197 75 165 307 0 6618 Sample Emnty 0 2 1 2 75 a7 71 203 5 IBJMFI 71 r 17 9 47 2d 8115 Sample Emnty 63 45 67 1351 5 io 53 8822 lWsiz2 Isampletmetv 60 aa 60 187 5 a 8 63 77 4 2 5012 Sample Emnty 48 38 51 191 3 47 4 5063 Samele Ent 1 1 1 1 1 O Samem Saray el Bog o Sample Empty 1 0 1 1 1 41 Sample Empty 3 1 2 2 2 6462 Sample Empty 2 0 1 2 2 6426 _ Sample Empty 2 0 1 2 1 4359s ample Empty 3 1 2 2 4 47 3d 6952 Sample Emoty i 4 3 3 gU 2 6188 ample Empty 2 1 4 3 3 v lili gt Figure 3 2 Typing table Bead names displayed in the order that the data were collected MasterPlex GT www miraibio com 3 3 CHAPTER 3 BEFORE YOU BEGIN Choosing a Bead Name Option You can choose between the two bead naming options that determine how the Typing table displays the results data 1 Select Option Set Application Options from the menu bar The Application Options dialog box opens Figure 3 3 plication Options Background Clustering Tool Plugins Bead Name Style Locus Name Allele Name C Original Bead Name Start Up Window After Data Loading S
6. L rAllele Call Parameters for IA 15124C Use Relative Intensity for Allele call Reportable Level 25 0 of total intensity Intensity Threshold ss MFI Intensity based Allele Call Call anything bigger than so MFI as an Allele Applv to all beads Figure B 2 Parameter Setting dialog box MasterPlex GT www miraibio com B 3 APPENDIX B PROJECT OPTIONS amp PARAMETERS B 2 Table View In the Table View tab Figure B 3 you can specify display defaults for the Typing table New Project Default Parameters Graph View Allele Cal View Custer Analysis Yalue To Show Adjusted MFI Cell Background Coloring Mode Q f Figure B 3 New Project Default parameters dialog box Table View tab Value to Show Make a selection from the drop down list to specifv the default Tvping table view that is displaved when vou open a results file csv or project gtp Choose from the following data formats e percent relative intensity e background adjusted MFI e bead count B 4 MasterPlex GT www miraibio com APPENDIXB PROJECT OPTIONS amp PARAMETERS Cell Background Coloring Mode 2 Tone Mode Gradation Mode IB Typing Sample2 Sample2 gtp Choose this option to use alternating colors to distinguish loci groups in the Typing table Figure B 4 Choose this option to use a color gradient to indicate relative percent intensitv of the alleles in a group Figure B 5 Jo
7. i C1 47 2 r C2 47 2 r C3 47 2d r G1 48 2 r G2 48 2 r G3 48 2d B2 47 1 r B3 47 1d F F3 48 1d B1 47 1 Fi 48 1 F2 48 1 I A1 beads only new H1 48 3 F H3 48 3d r H2 48 3 D2 47 3 r D3 47 3d r D1 47 3 C C4 beadsold gt E2 47 4 M E1 47 4 r A2 48 4 m Anderson Anderson 16126C Anderson 16126C 16126C Radd dd 4 XI XI lt lt 152C 152C 152C Anderson Anderson lt lt l lt lt lt 1 lt l Anderson KUKUK 1 Figure 7 18 Allele Call table Allele calls sorted bv expression level Allele calls the same as the reference are painted Allele Frequencv The allele frequency for a sample is Number of a particular allele call Total number of allele calls in the sample 1 To view allele frequency click the Allele Frequency tab in the Allele Call table Figure 7 19 2 To copy the allele frequency information right click the table and click Copy Table as Text from the shortcut menu that appears The table information is copied to the system clipboard 7 20 MasterPlex GT www miraibio com CHAPTER 7 RESULTS TABLES amp Allele Call Sample2 Sample2 gtp Bajas mm ij xl Allele Call Allele Frequency Genotype Frequency Haplotype Frequency Locus Allele Frequency IA 16124C 0 00 16126C 50 00 161294 0 00 Anderson 50 00 16217C 7 69 162
8. 0 6 12 Selecting a Group 0el 2o teatnausteadna 6 13 Importing a Group Set v4 0 ssrssmerzsnzazj 6 13 SAVING aA Projectes rerrer terido rrine Eiaa 6 14 Using the Save As Function 6 15 Opening a Projeti a rasa riss Baws awa Baws 6 15 CHAPTER 7 Results Tables The Typing Tables 8s oes ii ee cas ace 7 1 Viewing the Typing Table 7 1 Relative Intensity View 0 000 7 5 MPL VieW iii a ee sab ows 7 5 Bead Count View 0 000 ee eee 7 6 The Statistics Table si ienssnszese eto ejs 7 7 Sorting Samples in the Typing Table 7 9 Sorting by Sample Name 7 9 Sorting by Expression Level 7 9 Resetting the Sample Sort 7 10 Allele Call Table 06 7 11 Mer ins Wels 2 2 2 22tecescseneden eee 7 13 Allele Call Table Viewing Options 7 16 Aele PreQUen y 402015 ecu tie ina 7 20 Genotype Frequency s sess arei sarju 7 21 Haplotype Frequency Tida Homology Table and Chart 7 23 Viewing Graphs for Selected Samples 7 25 Multi Compare and Depth Bar Graph 7 25 Sample by Sample Scatter Graph 7 217 CHAPTER 8 Graphs The Multi Graph Views ssenjntisitetontewi 8 1 Sorting Samples by Expression Level 8 3 PCN ae anna tas Gade si ees aes ina 8 4 Multi Compare Graph 00 0 8 6 Depth Pa kein rr irei 8 8 Multi Compare and Depth Graph Display Options 8 10 Changin
9. 7 14 MasterPlex GT www miraibio com CHAPTER 7 RESULTS TABLES Well Merge Pos Sample Name Start Well rE B1 47 1 Color and group c1 47 2 number G indicate D1 47 3 the wells that will be Num of Well to Merge E1 47 4 merged in the Allele Ea GU faa Call table b d G1 48 2 H1 48 3 l Maximum Num of Yell Merge A2 48 4 B2 47 1 C2 47 2 D2 47 3 E2 47 4 F2 48 1 G2 48 2 H2 48 3 A3 48 4 B3 47 1d C3 47 2d D3 47 3d E3 47 4d F3 48 1d G3 48 2d H3 48 3d A4 4B 4d Cancel Figure 7 13 Well Merge dialog box Step 14 V Ignore Background 2 Inthe Start Well drop down list select the first well for the merge 3 Inthe Step box enter the total number of wells to include in the merge The group number well position and sample names included in the merge are updated Figure 7 13 4 Choose the Maximum Num of Well Merge option to create the maximum number of groups using the step number entered in step 3 5 Choose the Ignore Background option to omit background wells from the merge NOTE If the Background Hidden option is enabled the Ignore Background option is not available 6 Click OK The wells are merged in the Allele Call table Figure 7 12 7 To toggle the Allele Call table between the pre and post merge views click the Toggle Normal Merged Well button Tr MasterPlex GT www miraibio com 7 15 CHAPTER 7 RESULTS TABLES Allele Call Table Viewing Op
10. c MIT l Amel fa x Fi DYS199 oOo b DY 391 c 1 1 p 1 G MADT mal MEINA OCD ACE CoV a Ta Thea 2 4 ul Sample list Heat map Figure 8 2 Multi Graph view Heat map and Multi Compare bar graphs for three user selected samples 8 2 MasterPlex GT www miraibio com CHAPTER 8 GRAPHS Table 8 1 MasterPlex GT graphs Graph Type Multi Compare Graph Figure 8 2 Depth Graph Figure 8 7 Sample by Sample Scatter Graph Figure 8 15 Allele by Allele Scatter Graph Figure 8 17 Heat Map Figure 8 3 Displays a Bar graph of background adjusted median tluorescence intensity MFI or relative intensity RI values for each user selected sample Composite bar graph of background adjusted MFI or RI data for all user selected samples Scatter plot of background adjusted MFI data for a user selected pair of samples Each point represents an allele Scatter plot of background adjusted MFI data for user selected pairs of alleles Each point represents a sample Color coded representation of the MFI data for each sample Sorting Samples by Expression Level In the Multi Graph view you can sort the sample list by expression level MFI data This is useful for comparing and choosing samples for the graphs 1 Inthe sample list Figure 8 2 right click the sample you want to use as the reference for the sort 2 Click Sort By Expression in the shortcut menu that appears The
11. 7 To apply normalization to the selected standard at the associated allele MFI data click the entry in the Enable column and select TRUE from the drop down menu that appears Figure 10 21 If you do not want to normalize the data select FALSE 10 16 MasterPlexGT 9 www miraibio com CHAPTER 10 GENOTYPING USING A LOOKUP TABLE Lookup Table Editor Export Apply Reset All M4 8 Oor O ao Locus Setting Name Version 10 0 Group Name Reset Task f Standard Base MFI Cutoff Enable laoi lace laos face aos Jastdi JA sta Te A Stdi 1000 300 FALSE v TRUE FALSE Standard Beads Figure 10 21 Lookup Table Editor Standard Beads view Select TRUE from the drop down list to normalize the standard and associated allele MFI data If you do not want to normalize the data select FALSE 8 To associate alleles with the standard click the allele columns of interest Figure 10 22 Zl Lookup Table Editor Export Apply Reset All M 4 O8 Oor Oe Locus Setting Name Version 10 0 Group Name Reset Task f Standard Enable A 01 lace laos Jaos aos acstd JA staz v wf diin Type A Stdi TRUE v Standard Beads Cross Talk General Figure 10 22 Lookup Table Editor Standard Beads view Click the alleles that you want to associate with a standard 9 To reset all entries to the default value click Reset 10 To specify another stand
12. Inversion candidate or number of inversion candidates 10 To view results for another locus or the blood type click a tab Figure 10 41 11 To highlight the samples that exceed the MFI threshold click the Gradient Background button Results that exceed the MFI threshold are highlighted blue in the A locus tab red in the B locus tab and alternating red and blue in the remaining tabs Figure 10 42 10 32 MasterPlex GT www miraibio com CHAPTER 10 GENOTYPING USING A LOOKUP TABLE Bie zija a EO em eae oe SS tie mk re WellName JSample Name Total Events Notes _ Tvnera Am l PA a JIN MC TG TIT I al poooz heo T i ovans jj b jems a S ao Em s Ea a BCS Fa figooos_ fs faon aooga Es e1 figooog_sts2 fs aomo of i a ji 8 a2 figooos_ Jasas JI Junoni aoog Es 21 sa e a jamzama o c2 figooro Sst 7 PL fao s EW l D2 aoon fasan os oro of 17 E2 om2 sts Jaoos aor of 15 no pm s o a Isos laos aoo ie 10 42 Typing table gradient background The Typing table highlights MFI results that exceed the MFI threshold set in the Parameter Settings 12 To view the Allele Call window right click the Typing table and select Allele Call from the shortcut menu that appears The Allele Call window is displayed Figure 10 43 F Allele Call HLA A_B_DR_BLD a wa XI Allele Call Allele Frequency Genotype Frequency Haplotype Frequency A DR f B1 id0001
13. ee H9 hi6401 21 H8 hi6401 13 KUKU D8 h16401 9 C E7 h164012 C7 h16401 blank r C8 h16401 8 Samples FTABJKIGMIS C B7 h16401 blank lt l lt l 16223T Anderson 16362T 16223T 16311C 163627 lt l lt l with fewer r A4 h16236 blank called alleles than the reference Figure 7 16 Allele Call table Samples that have fewer alleles called at a locus than the reference sample display MasterPlex GT www miraibio com 7 17 CHA PTER 7 RESULTS TABLES Sorting Samples by Expression Level You can sort samples in the Allele Call table by expression level MFI data 1 7 18 In the sample column of the Allele Call table right click the sam ple you want to use as the reference sample for the sort and click Sort By Expression Level in the shortcut menu that appears Figure TAT The Allele Call table displays the reference sample in the top row and sorts the remaining samples by expression level in descending order top to bottom After the sort allele calls different from the reference sample are ainted highlighted with the group or allele color Figure 7 17 These colors are specified in the Parameter Setting dialog box see page 6 8 Allele calls that are the same as the reference sample are not painted MasterPlex GT www miraibio com CHAPTER 7 RESULTS TABLES FI Allele Call Sample2 Sample2 gtp sajas m palz x
14. Bead ave sp cv An amp Settings DER Ay File Edit View Favorites Tools Help Q ex Q JO search 1 Folders E ress O C Program Files HitachiSoft MasterPlex G 2 0 Settings v Go Size Type Date Modified 3KB FRFFile 10 1 2002 9 59 AM 3KB FRFFile 10 1 2002 9 59 AM 3KB FRFFile 10 1 2002 9 59 AM 3KB FRFFile 10 1 2002 10 00 AM 3KB FRFFile 10 1 2002 10 00 AM y Other Places ke Detail y y 3 KO PRPs TAGRA ae ERE 11KB GTTFile 10 31 2003 12 56 PM Ed Legal_L frf 3KB FRFFile 10 1 2002 10 00 AM Ed Legal _P frf 3KB FRFFile 10 1 2002 10 01 AM Ed Letter_L Frf 3KB FRFFile 10 1 2002 10 01 AM Ed Letter P frf 3KB FRFFile 10 1 2002 10 14 AM E MasterPlexGT gto 4KB GTOFile 10 31 2003 4 37 PM File and Folder Tasks Figure 10 3 Importing a lookup table To import a lookup table drag the gtt file to the MasterPlex GT application window or double click the file MasterPlex GT www miraibio com 10 3 CHAPTER 10 GENOTYPING USING A LOOKUP TABLE 4 To confirm the lookup table import a Open the Luminex results file csv or MasterPlex GT project gtp of interest b Click the Parameter Setting button fab The Parameter Setting dialog box appears Figure 10 4 Parameter Setting lt Group set RI Cancel Save setting as a Import Setting Parameter setup for the individual bead Minimum Events ko count for each bead
15. CHAPTER 3 BEFORE YOU BEGIN Haplotype for sample 47 1d Row of locus names FI Allele Call Sample2 Sample2 gtp Allele Call Allele Frequency Genotype Frequency Haplotype Freque is B2 47 1 V Anderson Anderson Anderson f B3 47 1d IV Anderson Anderson Anderson B1 47 1 Anderson Mama Anderson Anderson F3 48 1d V 16126C Anderson 16126C F1 48 1 IV 16126C Anderson 16126C F2 48 1 IV 16126C Anderson 16126C f G3 48 2d Anderson 162947 Anderson G2 48 2 Anderson 16294T Anderson G1 48 2 Anderson 16294T Anderson r C3 47 2d Anderson Anderson 16311C 16311C 16320T r C2 47 2 Anderson Anderson 16311C 16311C 16320T r C1 47 2 Anderson Anderson 16311C 16311C 16320T Sample name column Figure 3 13 Allele Call table displays the alleles called for each sample 8 SI Homology Plot Sample2 Homology Chart 0 46800 0 46368 0 40198 0 33525 0 32237 0 49930 0 49163 0 42308 035350 033786 0 32105 0 50343 0 49405 0 42395 035439 033874 0 32167 0 46246 0 45813 0 39433 0 32394 030967 0 29340 0 49154 0 48381 0 41243 0 33920 032233 0 30674 0 48291 0 47537 0 40571 0 33093 0 31525 0 29921 0 46800 0 49930 0 50343 0 46246 0 49154 0 49291 Sees 65208 0 65064 0 65860 0 46368 0 49163 0 49405 0 45813 0 49391 Te 0 67715 0 67667 0 68589 0 40198 0 42308 0 42395 0 39433 0 41243 0 40571 0 67401 0 67845 0 69039 0 33525 0 35350 0 35439 0 32394 0 33
16. CHAPTER 9 CLUSTER ANALYSIS idl JE Multi Compare Th Depth EE Threshold Editing EE Sample by Sample 4 La B fa Adjusted MFI of 5 F1 eft SOD i celal hail DARE Ceca ka 3 EL LIE a jp Oi KEE Ara ea a Q i E T L ARANA NASN ARNA i 0 a ta ata 1 nodna SNP1 SNP3 SNPS SNP SNPS Adjusted MFI of 8 42 SNP1 m t SampleSmall Clustering Tool N N Min F N Max Median Centroid a 8 mt LSNP2J B G Link Ward s Flexible ki Genotype Expression MFI Background 8 SNP1 SNP3 SNP5 SNP7 SNP9 Adjusted MFI of 4 E1 MFI Background Figure 9 3 Dendrogram in the MultiGraph view CODEN E Beads gt wt Well Name Total Events me a S E e2 fp Sf O E ss Fea JI KONI p FT SS Sn k C E B az Ba ft tog E Py es as es aa ed s non Ja SampleSmall Clustering Tool x NN Min F NMax Median Cel B G Link Ward s Flexible Genotype Expression Figure 9 4 Dendrogram in the Typing table 4 To select a different clustering tool click the tool name in the Clus tering Tool window The dendrogram is updated MasterPlex GT www miraibio com 9 3 CHAPTER 9 CLUSTER ANALYSIS 5 To close the dendrogram click the Close button amp in the Cluster ing Tool window 9 4 MasterPlex GT www miraibio com CHAPTER GENOTYPING USING A LOOKUP 1 0 TABLE The MasterPlex GT soft
17. Edit Selected Entry Opens the Lookup Table Editor for the selected table Create New Lookup Entry Opens the New Lookup Table Entry dialog box so that you can name a new lookup table Delete Selected Entry Deletes the selected lookup table Select Latest Table Selects the latest lookup table version for all loci Unselect All Clears the selection from the Lookup Table Selection window OK Accepts the lookup table selection and returns to the Parameter Setting dialog box Cancel Selects the default lookup table and returns to the parameter Setting dialog box MasterPlex GT www miraibio com 10 5 CHAPTER 10 GENOTYPING USING A LOOKUP TABLE 10 2 Creating a Lookup Table The Lookup Table Editor Figure 10 6 enables you to specify the table components including the Table name Type genotype name for the A B and DR loci and the blood type Type frequency Standards and allele expression patterns that define the type Display color for the type name in the MasterPlex GT Typing table a Lookup Table Editor E Apply Reset All O4 O8 Oor O eo Locus Setting Name HLA A version 11 1 Group Name Reset Ei iit Type Name Short Name Color Frequency lat Di A Stdi JA Std2 Type 4001 o0z 003 Standard Beads 004 005 006 007 008 General 009 010 Cross Talk Type A 009 allele expression pattern with a frequency 0 4 Figure 10 6 Lookup Table Editor Type task view 1 Open
18. IA IA Other 16124C 25 0 35 16126C 25 0 35 TM 16129A 25 036 35 Anderson 25 0 35 16217C 25 0 35 16223T 25 0 35 16224C 25 0 35 Anderson 25 0 35 Ici IC1 162927 16295T 25 0 35 16294T 25 0 35 Mi Group Allele Identifier Group Prefix of beads in this group 4 Edit Bead Names Group Name fia Change Color Ploidy Diploid Haploid Other Apply this Plaidy to all groups loci Apply to all alleles in the Allele Name Anderson Change Color same order in each group Apply to all same name alleles r Allele Call Parameters for 14 Anderson e Use Relative Intensity for Allele call Reportable Level 25 0 36 of total intensity Intensity Threshold 35 MFI C Intensity based Allele Call Call anything bigger than fo MFI as an Allele Apply to all beads Figure 3 4 Parameter Setting dialog box 2 Click Edit Bead Names The Edit Bead Names dialog box opens Figure 3 5 MasterPlex GT www miraibio com 3 5 CHAPTER 3 BEFORE YOU BEGIN Edit Bead Names Figure 3 5 Edit Bead Names dialog box 3 Select the bead name you want to edit and enter a new name 4 Click OK when you finish editing the names The new bead name prefix group and allele name is displayed in the Parameter Setting dialog box Typing table graph view and statistics table To rename a bead in the Typing table 1 Right click a locus or allele name in the
19. 25 0 35 25 0 35 SNP 25 0 35 M Group Allele Identifier Group Prefix F of beads in this group 2 tt Edit Bead Names Group Name enpr e Change Color Ploidy Diploid Haploid Other Apply this Ploidv to all groups loci Allele Name Allele Call Parameters for SNP1 e Use Relative Intensity for Allele call Reportable Level 25 0 of total intensity Intensity Threshold 35 MFI C Intensity based Allele Call Call anything bigger than 50 MFI as an Allele Apply to all groups loci MFI Allele Ratio Figure 6 12 Parameter Setting dialog box Group set drop down list displays available group sets Selecting a Group Set To select a group set for the active results click the Group set box and make a selection from the drop down list Importing a Group Set You can import a previously saved group set for example a group set created on another system 1 Click Import Setting The Open dialog box appears Figure 6 13 MasterPlex GT www miraibio com 6 13 CHAPTER 6 ALLELE CALL PARAMETERS Look in B Settings a Grouop Set 2 xml Group Set 1 xml Group Set 3 xml Files of type MasterPlex Setting File sml Cancel Figure 6 13 Open dialog box Group set files xml 2 Double click the group set file xml for import The Parameter Setting dialog box displays the imported group set 6 8 Saving a Project The contents of the Project Window c
20. 8 Jo Hi u Ble 8338535851 es pS pets zaa zac PT co E E 0 0 8 h16401 6 sooo Sample Empty 2 0 1 2 1 16401 12 4845 ae Empty Dz memi 6000 h16401 3 6000 a a amee Sort By Expression Cluster Analysis GenotvpejExpression Descending Open Bar Graph Open Scatter Plot Local Negative Control o o o ll p Figure 7 8 Typing table Sorting by sample name alpha numeric sort Sorting by Expression Level 1 Do either of the following to sort the Typing table by expression level Right click the sample name that you want to use as the reference for the sort and click Sort By Expression Level in the shortcut menu that appears Figure 7 9 Click the sample name that you want to use as the reference for the sort then click the Sort button The Typing table displays the reference sample in the first row and sorts the remaining samples rows by similar expression level in descending order MasterPlex GT www miraibio com 7 9 el leja F gt lal 4 ie X HEI Ii II IB Beats JiS124C Jis126C 161294 lAndersorji6z17C 162237 WellName Sample Name Total Events uE ET IT Fi jasi Is 110 86 132 100 88 66 Fz o Jea 124 4149 a9 122 66 152 132 160 1a2 112 77 a1 lbeads only new ki E E T z z B2 lt a 130 162 169 138 182 71 so 188 181 j 18770 97 FTI 2 T a taz 1007 1
21. C Use Relative Intensity for Allele call Reportable Level 50 of total intensity Intensity Threshold f 5 MFI e Intensity based Allele Call Call anything bigger than so MFI as an Allele Apply to all groups loci Figure 10 39 Parameter Setting dialog box 10 30 MasterPlex GT www miraibio com CHAPTER 10 GENOTYPING USING A LOOKUP TABLE Choose Intensity Based Allele Call set the MFI threshold to 50 and click Apply to all groups loci In the Parameter Setting dialog box click Lookup Table The Lookup Table Selection window appears Figure 10 40 The highlighted lookup table is applied to the results csv Lookup Table Selection Entry Mame Last Modified aati Selastad Erte PROJECT Create Mew Lookup Entry HLA Lookup Table 10 31 2003 12 56 50 PM HL4 4 HLAB HLADR Blood Type Yeri 10 VYer1 10 Veri l0 veri 00 Delete Selected Entry Select Latest Tables Unzelect All Cancel Figure 10 40 Lookup Table Selection window The window shows the lookup tables that are installed 6 8 Click the table that you want to use and click OK The software applies the highlighted table The newest lookup table version is the default Click OK to close the Parameter Setting dialog box The Typing table appears NOTE If a lookup table is not selected the Type column is empty To select a look up table open the Parameter Settings dialog box click ab and
22. GMP SNPG SMP SNPS SMP wk Adjusted MFI of 1 51 1 1 LI LI 1 LI LA l I LI LI LI LI ae 1 LI 1 1 1 1 1 LI 1 LI LI Liem pee ge ee re eee FE a ee ee taa i 1 er ee et tae 1 S uha Pait t a A E E AE T T T I 1 I I l I Oo ot l l l l 1 l EJ 1 l l l l I MEL T zi g I l l l l l l l I oo ae 1 1 1 if l l I I 1 l Ti L l l l EJ 1 I 1 l I l io jak Vi Wh Vi kol L w i L Vi ri ae Vi PUNDI HIEQ 14W Figure B 9 Multi Compare graph only called alleles painted top all alleles painted bottom www miraibio com MasterPlex GT B 10 APPENDIXB PROJECT OPTIONS amp PARAMETERS Adjusted MFI of 1 E1 110 105 100 e q4 r ee ee ee q47 r o U 1 L 1 1 1 r U 1 L 1 1 1 r 1 LI 1 L L LI 1 LI 1 LI 1 re rc 1 1 L 1 1 1 r 1 1 L 1 1 1 r MFI Background SNPS SMP SNPS SNPS Relative IntensityS of 1 B1 100 y 95 90 F BSE a0 F l ae eee E oR F ee ee YO F A 65 BOG F 55 F 50 F 45 Relative Intensity 40 ISBA 3095 25 20 15 10 so Os l E A SMPS SMP4 SNPS SAPS SMP SMPS SMPS SMP10 ie L SMP SMP2 Figure B 10 Multi Compare graph background adjusted MFI data top percent relative intensity data b
23. IC1 162927 1 15 41 7 ESTER IT 0 0 0 0 IC1 16294T 1 0 6 13 3 oOo oO o omma NENNI I Kn NHN B ORAO 1 PPN lt N NNG e NNG Ne Om no w e amp amp WH amp Ni a em aw a wD Figure 4 3 Project Manager docked to Project Window MasterPlex GT 3 www miraibio com 4 3 CHAPTER 4 GETTING STARTED Table 4 1 options Display Option Undock the Project Manager from Project windows Hide the Project Manager Tile Project windows in cascade Tile Project windows horizontally Tile Project windows vertically Minimize all project windows Organize all minimized project windows 4 4 MasterPlex GT Project manager and project window display Command Select View gt Dock Undock Project Manager from the menu bar or double click the Project Manager title bar Click the Project Manager Close button wi or select View gt Show Hide Project Manager from the menu bar Click the ka button or select Windows Cascade from the menu bar Click the button or select Windows gt Tile Horizontally from the menu bar Click the ITI button or select Windows Tile Vertically from the menu bar Windows gt Minimize All from the menu bar Windows Arrange All from the menu bar www miraibio com CHAPTER 4 GETTING STARTED Viewing Data The Project Window shows two views of the results data Typing table Figure 4 4 See The Typing Table on
24. Minimum Events ko count for each bead IV Use group color for Chart and Allele Call Table Lookup Table Lookup Table Allele Name Report Intensity Call Inten A Other HLA A v1 10 Other HLA B v1 10 lt i amp Group Allele Identifier Group Prefix of beads in this group 7 Edit Bead Names Group Name ja ETA Change Color Ploidy Diploid C Haploid Other Apply this Ploidy to all groups loci Allele Name mi r Allele Call Parameters for A Use Relative Intensity for Allele call Reportable Level 250 of total intensity Intensity Threshold MF Intensity based Allele Call Call anything bigger than bo MFI as an Allele Apply to all groups loci Figure 10 28 Parameter Setting dialog box 3 Click Lookup Table The Lookup Table Selection window appears Figure 10 29 10 22 MasterPlex GT www miraibio com CHAPTER 10 GENOTYPING USING A LOOKUP TABLE BI Lookup Table Selection T im Ed Entry Mame Last Modified Sef Sele Erte PROJECT HL44 HLAB HLADR Blood Type HLA Lookup Table 10 31 2003 12 56 50 PMEHL44 IHLAB HLA DR Blood Type Verl 10 Verdi 10 VYer1 10 Yeri 00 Delete Selected Entry Select Latest Tables Unzelect All Figure 10 29 Lookup Table Selection window 4 Click the table that you want to edit and click Edit Selected Entry The Lookup Table Editor displays the selected table Figure 10 30 FI Lookup Table Editor Apply
25. O Sample2 l Typing Table File ill Multi Graph O SampleBase tree Typing Table dl Multi Graph D SampleSmall Typing Table il Multi Graph 5 47 gt Figure 7 1 Project Manager To view the Typing table click the the project of interest Statistics table button under The Typing table displays the MFI and bead count data for the open results If the bead set names follow the MasterPlex GT naming convention the Typing table automatically organizes the data by loci and alleles Figure 7 2 See page 3 1 for more information about the bead naming convention If you merge results the Typing table displays all of the results See page 4 11 for more information on merging results 7 2 MasterPlex GT www miraibio com CHAPTER 7 RESULTS TABLES Locus group names first row and alleles names Second row m fo BIT Bla ul y Ras es a ee a ee ee Ss ee es ee es ee Beads Jis3osc Jiesiic lissiicmjiesisa 163207 landersorjissezc liesezr jissa lissamasliasc fisecnsof WellName JsampleName JTotalEvents nes S j jj a d S d d 11 5 pa mesma eooo JsampieEmotv 2 151 45 3 0 aj 3 sej i 2 1 1 27770141 a 1 si zaf 2 sp 2 o 1 1 145 a 9 z 15 10 s 0 a 0 1 1 65 14 as HEE aj a 1 2 1 2 1a 48 1 1 22 1o tos 0 1 1 1 F7 mesma feo 2 139 51 3 0 22 e gafo 1 2 1 a gg 2 33 32 9 0 iC o 9 1 0 HZ 2 3 1 83 2 A 65 3 0 1 0 0 BB mesz feo J S J e 2 1 86 0 2 8
26. Reset Alll O 4 8 Oor ear Locus Setting Name HLAA Version fa gf Group Name B Reset Task E Type Standard Beads Cross Talk General Figure 10 30 Lookup Table Editor 5 Click a locus tab MasterPlex GT www miraibio com 10 23 CHAPTER 10 GENOTYPING USING A LOOKUP TABLE 7 Click a task Type Standard Beads Cross talk or General To edit Refer to Type Defining a Type on page 10 9 Standard Beads Setting Standards on page 10 14 Cross Talk Specifying Cross talk on page 10 18 General Parameters Setting General Parameters on page 10 20 8 To return the settings in the current tab to the original values click Reset To return the settings in all tabs to the original values click Reset All 9 When you are finished editing the lookup table click Apply and click Yes in the confirmation message that appears Exporting a Lookup Table 1 Open the project gtp of interest 2 Click the Parameter Setting button Gb The Parameter Setting dialog box appears Figure 10 31 10 24 MasterPlex GT www miraibio com CHAPTER 10 GENOTYPING USING A LOOKUP TABLE FI Parameter Setting Group set bii ee ene gt Cancel Save setting as ll Import Setting Parameter setup for the individual bead Minimum Events fo o count for each bead IV Use group color for Chart and Allele Call Table Lookup Table mem loretan ire Lookup Table Allele Name Report Intensity C
27. prefix group and allele name is displayed in the Parameter Setting dialog box Typing table graph view and statistics table 6 6 MasterPlex GT www miraibio com CHAPTER 6 ALLELE CALL PARAMETERS Editing the Group or Allele Name Only You can also edit just the group or allele name in the Parameter Setting dialog box To edit the group name 1 Click the amp button to display the Parameter Setting dialog box Figure 6 5 Select the group name you want to edit Figure 6 5 3 Enter the new group name in the Group Name box and click Apply To edit the allele name 1 Select the allele name you want to edit Figure 6 6 2 Enter the new allele name in the Allele Name box Figure 6 6 and click Apply Parameter Setting DER Group set z Cancel Save setting as Import Setting Parameter setup for the individual bead Minimum Events ho count for each bead Use group color for Chart and Allele Call Table Lookup Table Prefix GroupName Type Lookup Table Allele Name Reportable Level Intensity Threshold Call Intensity A 35 ISNP1 SNP1 Diploid SNP2 SNP2 Diploid 0 35 SNP3 SNPS Diploid j 35 Group name SNP2 selected ISNP4 SNP4 Diploid 35 SNP5 SNPS Diploid j 35 w Group Allele Identifier l Group Prefix of heads in this group 2 tt Edit Bead Names Group Name ISNP2 Ploidy Diploid Haploid Other _Apply this Ploidy to all groups loci
28. slul je Al Jbesdsonivnew 2253 Sample Ematy 7787 Sample Empty SS Ss SS i eo ee Oo Beats fec Het 26 _ 161294 Jandersorjiozizc 162237 16224 _landersor 162927 til WellName Samole Name Total Events Notes IT L I a et es a ee Fi_ 4et_ s162_ samoetmoty Fi 88 9 10 E te f3 fasta desos Jsampietmiv 130 65 omeo ajoo Ca beadsold s1 Samne Ematy J Boo aa ws Sample Emnty 153 65 135 304 2 os Wia lesas Semle Emoty me Ee bmo laa 0 sma Serine Emoty 5 4 58 197 75 165 307 o 6618 Sample Empty 1 2 75 4 71 203 5 a o wa 8115 Samnle Emptv 2 2 69 a5 er 185 5 ao e2 laz Sample Empty 1 1 60 44 60 187 5 cea Jas 2a sm2 Samvle Ematv 1 E 47 4 5083 JSampleEmetv 1 1 1 1 1 1 E2 la7 g 3109 Samoe Emoty 0 0 2 i IT 0 1 1 JIM ao o gpg Jezat Sample Empty 0 1 1 2 3 1 2 2 EB a e462 Same Ematy 2 2 2 0 1 2 ae As 8d 6426 Samnle Emptv 1 1 2 a 2 0 1 z uo Wwa laa Sample Empty 1 2 xi 3 3 1 2 2 4 Do o la7 3d ea52 Jsampie Ematy 0 2 2 1 3 3 1 2 mo lea lms Sample Empty 2 2 1 3 2 1 4 3 hal Figure B 4 Typing table two tone mode stripe background MasterPlex GT www miraibio com B 5 APPENDIX B PROJECT OPTIONS amp PARAMETERS zz TTIE IE en pe See eee Beats 6124 16126C 161298 Jandersorjiszizc J162231 l16224C landersor WellName _ SampleName _ TotalEvents notes doood S S S S S Fo assy 5286 Sample Emet E3 asta Jesos Sample Emoty Al
29. 2 Installing MasterPlex GT IREGUMCWNEMIG nt seuss a 2 1 Installing MasterPlexk GT 2 1 Tastaling a Licens lt cese setsctecevays reta Zo CHAPTER 3 Before You Begin Bead Name Conventions 00 3 1 Choosing a Bead Name Option 3 4 Editing the Bead Name 3 4 Overview of MasterPlex GT Analysis 3 8 CHAPTER 4 Getting Started The Project Manager and Project Window 4 1 Viewing Project Information 4 7 Removing Projects from the Project Manager 4 7 Opening Luminex Results 000 4 8 Opening Results Using the Menu Bar or Toolbar 4 8 Merging Results sssssdi etjntonez joni Qas 4 11 sample MEGS aston sich ie ita alik 4 11 Layer INICIO 2 chet ce hehee ced etaea 5 oun 4 15 Editing a Bead Name s sspsisdestedzejjon 4 19 CHAPTER 5 Negative Controls Local and Global Negative Controls 5 1 Setting Negative Controls Manuallv 5 2 Setting Negative Controls Automatically 5 3 MasterPlex GT www miraibio com Vil CHAPTER 6 Allele Call Parameters Parameter Settings and Options 6 1 PIO SE 6 3 CONTENTS Relative Intensity Allele Call 6 3 Intensity Based Allele Call 6 5 Editing a Bead Name 4 6 6 Group and Allele Colofsscac6 ius abueonnegas 6 8 Changing the Group or Allele Color 6 9 Working With Group Sets 0 6 12 Creating a Group Set
30. 35 73G sl 934 Z 93G g Page178 al PE gt Figure 11 4 Report Preview window 2 To scale the view click the scale toolbar button T 1 and choose a view option 3 To close the Preview window click the Close button Searching a Report In the Preview window you can perform a text search in the report 1 Inthe Preview window click the Find text button i The Find text dialog box appears Figure 11 5 Find text Text to find moo Options Origin Case sensitive 1st page Curent page Figure 11 5 Find Text dialog box 2 Enter the text string for the search 11 4 MasterPlex GT www miraibio com CHAPTER 11 REPORTS 3 If necessary choose the Case sensitive option 4 Choose an Origin option for the search 1st page starts the search at the first page of the report or Current page starts the search at the page currently displayed in the Preview window 5 Click OK to start the search Saving a Report 1 Inthe Preview window click the Save toolbar button mi The Save As dialog box appears Figure 11 6 Save in O SampleData Save as type Report file frp vi Cancel Figure 11 6 Save As dialog box 2 Select a directory and enter a name for the report rpt 3 Click Save Printing a Report 1 Inthe Preview window click the Print toolbar button J The Print dialog box appears Figure 11 7 MasterPlex GT www miraibio com 11 5 CHAPTER 11 REPORTS P
31. B2 47 1 T B3 47 1d Bi 47 1 Anderson Anderson Anderson Anderson Anderson 62176 Anderson lt lt l lt l C1 47 2 xl Anderson Anderson 16311C 16311C 16320T Anderson Anderson 16311C 16311C 16320T Anderson Anderson 16311C 16311C 16320T Anderson 16294T Anderson Anderson 16294T Anderson Anderson 16294T Anderson C2 47 2 KU C3 47 2d xl r G1 48 2 G2 48 2 r G3 48 2d r E1 47 4 r a2 48 4 r E2 47 4 r a3 48 4 r E3 47 4d lt u Kra Allele Call Sample2 Sample2 gtp El feo ro ma x Allele Call Allele Frequency Genotype Frequency Haplotype Frequency C A1 beads only new Faron Anderson 16217C Anderson 16311C 16362T 734 93G Anderson 195C 263G Anderson 16311C 16320T Cee 16126C Fi 162941 Anderson 163621 73G 934 1526 195C 263G C 3147 M Anderson Anderson Anderson 16311C 16362T 734 93G Anderson 195C 263G 16311C 16320T M M IA IB ICI Ic2 ID IAI JIIAZ IIB IIC IID r 4 48 16126C parergon 16294T Anderson 16362T 73G 938A B 195C 263G r 5 47 d Anderson Anderson Anderson 16311C 16362T 734 93G Anderson 195C 263G 16311C 16320T 6 48 d_M 16126C i 16294T Anderson 16362T 73G A B 195C 263G 4 Figure 7 12 Allele Call table before a well merge top and after a well merge bottom 1 Inthe Allele Call table click the Open Well Merge button ae The Well Merge dialog box appears Figure 7 13
32. HAZ IE 162947 ti Figure 8 6 Multi Compare graphs Relative intensity data 8 4 Depth Graph The Depth graph plots the expression profiles background adjusted MFI or RI data for user selected samples in one bar graph It is a useful way to compare allele expression levels across samples 1 Open the Multi Graph view for the results you want to graph and click the Depth tab 2 Inthe Sample Name list Figure 8 7 highlight the samples you want to display in the Depth graph To select adjacent samples press and hold the Shift key while you click the first and last sample in the selection To select nonadjacent 8 8 MasterPlex GT www miraibio com CHAPTER 8 GRAPHS samples press and hold the Control key while you click the samples The Depth graph displays the background adjusted MFI data for the selected samples Figure 8 7 To display allele information put the mouse pointer over a bar A pop up tool tip displays the allele name and intensity data Figure 8 7 To display relative intensity RI data Figure 8 8 click the RI button To rotate the 3 dimensional 3D view of the Depth graph click and hold the mouse while you move the mouse pointer in a hori zontal or vertical direction The graph view rotates horizontally or vertically To reset the 3D view right click the graph and select Reset 3D View in the shortcut menu that appears To clear the Depth graph click an empty row in the sample
33. Hypervariable Region IA fea Sea Parameter Settings enter IA 16124C IA 16124C or IA 16124C IA 16126C IA 16126C or IA 16126C IA 16129A IA 16129A or IA 16129A IA Anderson IA Anderson or IA Anderson 3 2 MasterPlex GT www miraibio com CHAPTER 3 BEFORE YOU BEGIN First row shows locus or group name the prefix set in the Luminex software Second row shows allele names at each locus IBI Typing Sample2 Sample2 gtp a xf 2 sj i II Ce es Fes eae ed Beats fst 24 16126 161298 andersor 16217 _ 16223T_ 16224C_ Andersor 16292T 11 WellName SamoeName TotaiEvents notes TP ao Jea 5162 Sample Emoty i F3 oea la Semple Emoty f3 fata esos Jsampietmatv Al beads onlvnew 2253 Sample Emoty ca beadsold 951 Sample Emoty B2 fare sts Semi Emoty Bs Waa lesas Sami Emoty bmo aa 19 SamoeEmty c2 fag eog Sami Emoty 47 2d 8115 Sample Empty 5122 Sample Emetv e jez Jsiss Samle Emoty er aa 5063 SamoeEmoty a2 aaa ft JSampletmatv eg Jara 109 Sample Emoty E3 araa 462 JsampieEmotv ore Jaza assa Sample Emoty D Wwa l D aad s952 Sample Emoty TE 48 3 6188 Sample Emptw K Figure 3 1 Typing table Bead names sorted by prefix and allele name a Jo ET gt u ale a II IN PI i IB Anders I Beats Jin 181 24 14 161 26 14 16129414 Andersjib 16217EJIB 162237 16224 WellName _ SampleName TotaiEvents notes TT 305 3
34. MFI ia Count l Heat map SNFI mt SNP2 wt o o di 00 i o on Oo o Statistics table SNPS mt SNP1 wt SNP wt SNP10 mt Figure 8 3 Heat map Alleles from left to right 8 4 MasterPlex GT www miraibio com CHAPTER 8 GRAPHS 1 To show or hide the Heat map click the INI toolbar button To view allele MFI data position the mouse pointer over the allele of interest in the Heat map A pop up tool tip shows the allele name background adjusted MFI data and relative intensity data Figure 8 3 3 To change the width of the bars in the map open the Applications Options dialog box select Option Set Application Options from the menu bar and enter a pixel number for the bar size min imum 1 pixel allele maximum 10 pixels allele Figure 8 4 BI Application Options Bead Name Style f Locus Name Allele Mame Original Bead Mame Start Up Window After Data Loading i Show Table view C Show Graph View Table View Gradation Background Use Allele Call Color far gradation background Group Color 1 IMMA Change Color Group Color 2 7 Change Color Heatmap Options Heatmap Bar Size j3 Pixel s f Allele Reset All Cancel Figure 8 4 Application Options dialog box MasterPlex GT www miraibio com 8 5 CHAPTER 8 GRAPHS 8 3 Multi Compare Graph The Multi Compare bar graph displays the background adjusted MFI or RI data for user selected samples in a
35. Sample by Sample scatter plot 8 20 MasterPlex GT www miraibio com CHAPTER 8 GRAPHS 7 R2 0 69039207 Seppe ka ad Pee es pee ef Sea Se See a a a ee a aw ee a we eae a ee alale oe ele o SS SASS A SS SSS SSS SSS eee SAS IA A A e MN Ne NaN Merk ser Mao MMe Ni i ben BN tM Men lenin siekta Nitta mina algi belie She ase eae nso ae ee Ee SS eee Slee eae ele eee lh B Mo eS aa se om oe Ea IC2 16311C ad ec cae iTTAMENSONn ee ee Ls oe ee oe Faw ee je os we oe me oe re ee ae eo et ae eo pes 130 i i i i i i i A 120 J PUA SAAN 4 TE EPEN DATA DENE GESA LL EVAAA Ee EEE REE R ER a sa SS TSA N ee EEE AE 1 i I i l Semen C2 16311C 163207 Sees Ee eee nine See poe eee 2 Lin sot 3 i i E gma ee 5 A A era ee A ns d s 18 p p fp p p p r 7 7 banana ERETI ETET Sr ds E TE E aa Mis E L EE ep E et EE epe a 2 40 60 B0 100 120 140 ded ina 200 Arliniztori MFI nf 48 29 BTi Figure 8 16 Sample bv Sample scatter plot Allele labels displaved 8 8 Allele bv Allele Scatter Graph The Allele bv Allele scatter graph plots the MFI data for two user selected alleles from all samples in the active results The scatter graph distinguishes between samples in which both alleles are called neither allele is called only the x axis allele is called or only the y axis allele is called 1 Open the Multi Graph view for the results you want to graph
36. To specify a paper size and orientation for the report make a selec tion from the Paper Size Orientation drop down list The paper size and orientation is set for the report options sample information cluster analysis heat map allele calls charts and raw data 5 To select the paper orientation for a single report option click the Portrait or Landscape radio button for the option of interest For example in Figure 11 2 landscape orientation is chosen for the raw data only Report Manager Sample by Sample Scatter h408 1 vs h16236 o Sample Information Allele by Allele Scatter IA 16126C vs IB 1622 iv Include Sample Information to Report IV Include Cluster Analysis to report IV Instrument Status IV Background Information iv WV Allele Call Parameter Settings Porta C Landscape e Portrait Landscape IM Include Allele Call Tables to report F Include Raw Data to report V Allele Call Table Allele Frequency Table IV Genotype Frequency Table IV Haplotype Frequency Table e Portrait C Landscape lv Include Charts to report Paper Size Orientation LETTER Portrait e Portrait C Landscape Preview Report Save Settings As Default Close Landscape orientation selected for raw data only Figure 11 2 Report Manager 6 To save the selected report options as the default click Save Set tings As Default Adding Charts to a Report 1 In the graph view click the Wil toolbar bu
37. Type Ploidy Unmatch Caption No Matches Comma Split TRUE Standard Beads Include Standard TRUE Cross Talk General Figure 10 27 Lookup Table Editor General view Parameter Description Ploidy Choose diploid haploid or other Unmatch Caption The default unmatch caption is No Matches The Typing table displays No Matches when there is no genotype match for a sample The caption is user editable Comma Split Describes how the Typing table displays a geno type The comma split display for example A 002 A 010 is the only option available at this time Include Standard TRUE include standard data in the Typing table FALSE do not include standard data in the Typ ing table 3 To select a ploidy option click the ploidy value and make a selec tion from the drop down list that appears diploid haploid or other 4 To edit the unmatch caption double click the value and enter a new value MasterPlex GT www miraibio com 10 21 CHAPTER 10 GENOTYPING USING A LOOKUP TABLE 10 3 Managing Lookup Tables You can edit export copy or delete a lookup table Editing a Lookup Table 1 Open the Luminex results csv or project gtp of interest 2 Click the Parameter Setting button GP The Parameter Setting dialog box appears Figure 10 28 BI Parameter Setting Group set peer ayaa Cancel Save setting as Import Setting Parameter setup for the individual bead
38. USING A LOOKUP TABLE Jo GEIS a E0 8 pr ja n wf fet ft o Sta Jsta2 WellName JSampleName Total Events Notes Tvoermn jam v Eji a a TAW AT giches T TE Bi fidooot fs fBoo Boo3 2f 15 jg g O a IG IO AI at ft SS a a po 8 001 02 lt __ _ _ aa as ee ee EE ee ee A S INI ee KI gg l I jim l OI EG EEI jg eee ee Eee i i ENRI 7 EE ee ee eee L es ee jp L L aooe E pe T E a A o eee l OI jet fiaooo2 fasso tf Boos sf 20 Di fioa fara TK Bor SP 27 E1 Jioooa faro faon of 5 8 Fa fidooos_ sos St No Matches of 3 lot Jiooos Jsisz 777 1 r Boon Hi fiaoooz fssso sf fe Boro s 27 a2 fiaooog fasas Boo Boos F 1 B2 fog fssmn f Mo Matenes 0f 2 eaaa eee Eee A eee so hmn af pus jaooo fasst oot soo e foot lene fo os 10nd E2 ignores S fr Boos f 8 20 o Beads gt Genotype No Matches call B 001 B 003 A i esiastanssntsansnnadi B 001 is an inversion candidate because the genotype is a possible call if the MFI B 01 lt threshold B 007 is an inversion candidate because the genotype is a possible call if the MFI B 02 is lt threshold Figure 10 46 Typing table MasterPlex GT www miraibio com 10 37 CHAPTER 10 GENOTYPING USING A LOOKUP TABLE 10 38 MasterPlex GT www miraibio com CHAPTER REPO RTS 1 This chapter explains how to Gene
39. and click the Allele by Allele tab 2 Inthe allele list Figure 8 17 press and hold the Shift key while you click the two alleles for the scatter graph The Allele by Allele scatter graph for the user selected alleles is displayed Figure 8 17 MasterPlex GT www miraibio com 8 21 CHAPTER 8 GRAPHS G SRY 465 C SRY 1532 A G SRY 3225 C T sY814 G TatC ili Max 100 AUTO Allele list Alleles not called in x axis intensitv v axis intensitv either sample threshold threshold Figure 8 17 Allele bv Allele scatter graph The graph points are identified by color Graph Point Color Represents a sample in which White Neither allele is called Red default Only the x axis allele is called Blue default Only the y axis allele is called Black Both alleles are called NOTE You can change the red and blue default colors in the Allele by Allele scatter graph for intensity thresholds and graph points in the Application Options dialog box See Changing the Gradient Background Colors on page A 3 Ii 8 22 MasterPlex GT www miraibio com CHAPTER 8 GRAPHS 8 9 Copying a Graph To copy a graph right click the graph and select one of the following from the shortcut menu that appears Copy asa Bitmap Copies the graph in bitmap format bmp to the system clipboard Copy as Windows Copies the graph in Windows metaformat emf MetaFormat to the system clipboard Copy All Charts as Copies all graph
40. and move the slider to the right or left A 4 Plug ins This tab shows plug in applications that are available to the MasterPlex GT software 1 Select Option Set Application Options from the menu bar The Application Options dialog box opens Figure A 12 a Application Options General Background Clustering Tool Plugins Plugins Selection Reset All Cancel Figure A 12 Application Options Plug in tab 2 Click the Plug in tab 3 Place a check mark next to the plug in application that you want to use with the MasterPlex GT software The plug in o automatically starts the next time MasterPlex GI is started MasterPlex GT www miraibio com A 9 APPENDIX A APPLICATION OPTIONS 4 To disable a plug in application remove the check mark next to the it A 5 Resetting the Application Options To return all user modifiable settings to the factorv set defaults click Reset All Figure A 12 A 10 MasterPlex GT www miraibio com APPENDIX PROJECT OPTIONS amp PARAMETERS The project options and user modifiable parameters are the default settings that the MasterPlex GT software applies when you open a results file s csv and start a new project You can set defaults for the allele calling algorithm Typing table view Multi Compare and Depth bar graph display Allele Call table view cluster analysis tool These settings apply only to new projects This appendix explains the types of op
41. bar graph format It is a useful way to view the sample genotype or haplotype and compare expression levels across samples 1 2 8 6 Open the Multi Graph view for the results vou want to graph In the Sample Name list highlight each sample that vou want to displav in a Multi Compare graph Figure 8 5 To select adjacent samples press and hold the Shift kev while vou click the first and last sample in the selection To select nonadjacent samples press and hold the Control kev while vou click the samples A Multi Compare graph displays background adjusted MFI data for each selected sample Figure 8 5 To display relative intensity RI data Figure 8 6 click the RI but ton To display allele information put the mouse pointer over a bar A pop up tool tip displays the allele name and intensity data Figure 8 5 To clear the Multi Compare bar graphs click an empty row in the sample list MasterPlex GT www miraibio com Pos Sample Name B3 47 1d B2 47 1 F3 48 1d F1 48 1 G3 48 2d G1 48 2 C3 47 2d C2 47 2 Ci 47 2 C4 beadsold B4 beads new Hi 48 3 H3 48 3d H2 48 3 D2 47 3 D3 A47 3d Di 47 3 E1 47 4 A2 46 4 E3 47 4d A4 48 4d E2 47 4 48 4 beads only new CHAPTER 8 GRAPHS idl EE Multi Compare Ty Depth EE Threshold Editing Sample by Sample_4 gt EO AI Feat Some IS E OS Geet Wins E tet Vee TT E S fea Vee eT TR TIFI PA Soret Dold Veet T O E TATI Fi
42. csv file that you want to open 3 Select the csv file then click and hold the mouse button while you drag the selected file to the MasterPlex GT application window Figure 4 9 4 Release the mouse button gt The csv file opens in MasterPlex GT The Project Manager and Project Window appear Figure 4 8 In the Project Manager the file tree displays the file name The Project Window displays the Typing table default MasterPlex GT File Edit View Option Window Help Delma x Sales Hull qlalej SE r fis SampleData Sele a File Edit View Favorites Tools Help Q sxx z JO search fey Folde Address C Program Files HitachiSoft MasterPlex GT 2 0 SampleData v Go Folders x Name Size Type a MasterPlex GT 2 0 A JEJHLA Lookup Tabl att 11KB GTT File J O SampleData J JHLA A B DR BLI csv 7KB Microsoft Excel Comma Separated Values File Settings us HLA A B DR BLDjatp 46KB GTPFile MasterPlex QT 2 0 J LaverSampleA csv 35KB Microsoft Excel Comma Separated Values File E HTML Help Workshop 3S LayerSamplec csv 35KB Microsoft Excel Comma Separated Values File B InstallShield Installation vi MS JLayerSampleG csv 35KB Microsoft Excel Comma Separated Values File v lt u gt i lil Fl Figure 4 9 MasterPlex GT application window and Windows Explorer To open a Luminex results file drag the file
43. data 1 To create negative controls in the Typing table click the names of the samples that you want to designate negative controls There are three ways to do this Click a sample name To select adjacent sample names click and hold the mouse while you move the pointer over the sample names Click the mouse when the complete selection is highlighted Alternatively press and hold the Shift key while you click the first and last sample name in the selection To select nonadjacent samples press and hold the Ctrl key while you click the sample names The selected sample rows are highlighted 5 2 MasterPlex GT www miraibio com CHAPTER 5 NEGATIVE CONTROLS 2 To set the selected samples as negative controls right click a high lighted sample name and do either of the following If you are working with one results file click Local Negative Con trol in the shortcut menu that appears If you are working with merged results click Global Negative Control in the shortcut menu that appears The selected samples rows are designated negative controls and the rows display dash marks Figure 5 1 jo El HE ll al o toc sept spn sues een fet fat ft ot ft WellName JSampieName Total Events notes omde d l d TAW KT 7 354 21 475 39 656 43 307 291 548 40 913 35 403 35 702 106 1244 134 605 4a 725 645 368 68 50 of 50 8l 45 21 450 33 567 37 700 80 334 31 579 ar 967 1
44. dialog box appears Figure 10 19 New StandardBead Name Entry StandardBead Name A_Std1 X om Figure 10 19 New Standard Bead Name Entry dialog box 4 Click the drop down arrow and make a selection from the drop down list and click OK The standard is added to the Lookup Table Editor Figure 10 20 MasterPlex GT www miraibio com 10 15 CHAPTER 10 GENOTYPING USING A LOOKUP TABLE E Lookup Table Editor Export Apply Reset All M 4 O8 Oor Oa Locus Setting Name version 10 0 Group Name Reset Standard Base MFI Cutoff Enable lao Jao laos Jaos laos Ja stdi JA std2 TE A_Stdi MI 300 FALSE Standard Beads Figure 10 20 Lookup Table Editor Standard Beads view Confirm the default base MFI and cutoff values or enter new values 5 Confirm the default base MFI value or enter a new value The software uses the base MFI value to normalize the MFI values of the alleles that are associated with the standard To normalize the data the software sets the standard MFI equal to the Base MFI and computes Normalized Allele MFI Allele MFI x Base MFI Standard MFI oF NOTE The cutoff value must be less than the base MFI value 6 Confirm the default cutoff value or enter a new value If the standard MFI is less than the cutoff value the software does not normalize the standard or allele MFI data NOTE The cutoff value must be less than the base MFI and greater than one
45. dil Multi Graph Figure 4 15 Project Manager MasterPlex GT www miraibio com 4 15 CHAPTER 4 GETTING STARTED Project Merge Wizard Sample Merge Merge Some projects with the same bead set Layer Merge Merge Some projects with the same Wells Figure 4 16 Project Merge Wizard 2 Click the Layer Merge button The wizard displays a drop down list of results open projects Figure 4 17 4 16 MasterPlex GT www miraibio com CHAPTER 4 GETTING STARTED Make a selection from Open projects that can be layer merged with the the list of open projects project selected above If you do not want to include a project in the merge remove the check mark Layer Merge Wizard E BE Locus Beads Project Locus A LayerSamples LayerSamplec LayerSamplec e Layersamples G LayerSampleG LayersSampleT T LaverSaripleT Locus Top Project Layersampler LaversampleA LayerSamplec LayerSampleGg LayerSampleT LayerSamples LayerSamplec LayerSampleG LaversampleT LaversampleA LayerSamplec LayerSamplec LayerSampleT LocusS i Delete Project for Merge A LayerSampleA LayerSamplec jil ok Accept Cancel G LayerSampleG T LayerSampleT Lo Ld Choose this option to Bead list Project names remove all but the top organizes project name from the names by Project Manager after locus group the merge name Figure 4 17 Layer Merge Wizard 3 Make a selection from the Top Proje
46. list idl JEJ Multi Compare Th Depth Threshold Editing EE Sample by Sample Allele 4 d SampleSmall wt _ _ G Y Y a w Q H Sasso O amp O os D O o 0O amp MFI Background oe A Q y Q Wi o o o m o o o O o hd i Oo o Jdlldadd mt SNP2 SNPS SNP4 SNPS SNPS SNP SNPS SNPSSNP10 kan Figure 8 7 Depth graph Background adjusted MFI data for samples 5 6 7 and 8 MasterPlex GT www miraibio com 8 9 CHAPTER 8 GRAPHS TEE Multi Compare Gh Depth EE Threshold Editing E Sample by Sample Allele by Allele Samples mall a E e Sete E E E E E craves Serer a LSHF1J wt MA e JPN mit 1 700 SMP2 of bene eee ar r 7 9 EA 4 3 ust 4 600 4 mt WAWA IED SNPS 1 500 4 wt r E a mt 1 400 4 SNPS a eae ee ee eK He He Hee 06 A A a a a Baa wt 1 300 4 mit ee eee eee ee SNPS 4 200 4 wt lt bee eee eens ap bee ee nn Fo oe oo re mit 1 100 SNPE E o p Teg BB i Mai L I wt 1 000 4 mit G f F MI F oE CMER Il 4 SNP 3 a00 L oot oO Lowe 0 MM A ME E MU l M SS a mt wo isa goo l SMPS L eee ee lee E MS MM bl ut Foot mit SNP wt Boo soo 4 SNP10 oo M 300 ooo d 100 4 JU ing i mi Deg Ligi z a z u G GSNFI SNMP2 SNPS SMP4 SMPS SHPE SMP SNPS SNPS
47. name the new group color is displayed in the Group Name color swatch and is applied to the data for the alleles of this group in the Multi Compare Depth graph and Allele Call table If you selected an allele name the new allele color is displayed in the Allele Name color swatch and is applied to the data for the selected allele in the Multi Compare Depth graph and Allele Call table 6 7 Working With Group Sets You can save the settings in the Parameter Setting dialog box Figure 6 12 as a group set xml for use in future sessions You may also import a previously saved group set Creating a Group Set 1 Open the Parameter Setting dialog box click the 18 button 2 Click Save setting as The Create a New Group Set dialog box appears Figure 6 11 Create a New Group Set New Group Set Name SS Figure 6 11 Create a New Group Set dialog box 3 Enter a name for the group set and click OK The group set xml is saved and the name is added to the New Group Set name drop down list Figure 6 12 6 12 MasterPlex GT www miraibio com CHAPTER 6 ALLELE CALL PARAMETERS TI Parameter Setting SEE Group set Group Set 1 Group Set 1 Cancel Group Set 2 Parama roup Set 3 um Events so count for each bead IV Use group color for Chart and Allele Call Table Lookup Table Lookup Table Allele Name Reportable Level Intensity Threshold Call Intensity A SNPL SNPI Diploid 25 0 35 25 0 35
48. name on the bottom axis of the graph Display a subset of the labels so that none overlap on the bottom axis of the graph Changing the Y Axis Maximum 1 left of the graph Figure 8 9 GRAPHS Click RI I t ie To change the maximum of the y axis scale move the slider at the The graph is updated using the new y axis maximum the status bar displays the y axis maximum For example the Multi Compare graphs in Figure 8 9 plot the same samples using different y axis maxima MasterPlex GT www miraibio com To reset the y axis maximum to the default click the Hl button 8 11 CHAPTER 8 GRAPHS y axis slider hi JEF Multi Compare h Depth EE Threshold Editing Sample by Sample E Allele by Allele Anderson Adjusted MFI of 47 1 B1 1A 16124C 16126C 16129A Anderson See ee eee 1B mm 16217C 197 16223T 75 16224C o o N b o 1 1 1 a ll EN TUE 1 1 T o o ao Fe 5 i etal xi S MLM ML l Ic2 ID 181 142 IB IC ID MFI Background o Adjusted MFI of 48 2 G1 IA 3 Se SS a aaa a 16124C S 300 Andersona i 16362T i i 16126C 5 RT dget SE E es e ATA l Kurat 3 16129A 200 i i i Anderson h h i Anderson amp ere le Rae ata Tama i IB a 100 k LI ki Sia tei 16217 Ss ola 7 az LA A ME LLL LM A iew IA ID 181 IA2 IIB ic Ip Adjusted MFI of 47 4 E2 S00 ff 2 aoe amie
49. name tags are rotated in a counter clockwise direction Allele name tag slider Multi Compare TH pE Threshold Editing BE G Sample by MTA ke Allele by Allele Adjusted MFI of 45 2 61 161240 1 161260 2 161294 i Anderson 1 IE 162170 61 16223T 44 162240 Anderson 187 Ici 162927 16295T EME MELEE ELE EME EE EE IA ID WAT WA IB lZ ID 1G294T 110 Figure 8 10 Multi Compare graph Rotated allele name tags MasterPlex GT www miraibio com 8 13 CHAPTER 8 GRAPHS 2 To manually reposition a name tag click and hold the tag and move it to a new position Magnifying the Graph You can magnify or zoom in on a user selected area of the graph 1 To zoom in on the graph click and hold the mouse while you draw a rectangle from upper left to lower right corner over the area of interest Figure 8 11 The Project Window displays the selected graph area Figure 8 12 2 To zoom out and return to the original magnification click and hold the mouse while you draw a rectangle from right to left in the graph The Project Window displays the graph at the original magnification Adjusted MFI of 2 C1 Baller fee 800 750 700 MFI Background 350 300 250 200 150 100 50 l 4 SNP7 wt SNPS mt SNP10 wt 0 4 A i SNP1 wt SNP2 mt SNP4 wt SNPS mt Figure 8 11 Multi Compare graph Rectangle specifies the area to be magnified 8 14 Ma
50. of the allele name tags are displayed in the graph Figure 8 16 7 To plot aa new scatter graph that includes the sample at the top of the sample list click another sample The Sample by Sample scatter graph for the two samples is displayed Pos Sample Name HEE Multi Compare Th Depth EE Threshold Editing E Sample by Sample EE Allele by Allele AL beads only new l BI 474 C1 472 48 2 vs 47 2 R2 0 67845133 DI 47 3 a PAP sara PPR POTEET OEI i l i E1 47 4 4 190 f i rigs cal apnea i FE e a jesa FI 48 1 180 t 4 pe corer eee pongo nee B aaa MA AM JAG ALU eee eka E Agia uaa AR H1 48 3 L A2 49 4 160 er ba ke a Rs I a a a a a Se ak aie Il ee Ee a i aa ea a i ata X axIS B2 47 1 150 sample Ma ue senent IPA TIRI a a G ATA MAR MEStEOE S A D2473 Jl e A el E2 47 4 A jesa axis F2 484 120 y 49 2 a LT care aa ak a ses ese a sample H2 48 3 II A PM MAP MOM PTE EE ETT 3 48 4 TREE PATH RARAAL CU NAAT TASAL TST E STANTE L ETT ePeetl esi ersys B BDL B3 471d 63 EF L I Mar a za a A cca e A D3 47 3d A d dalma vais ata jedd i a etch chee iota cio E3 47 4d SCE A A DA F3 48 1d 50 MA a a ge a a a ca a l G3 48 2d H3 48 3d Sila ee Aaa a ces oa aaa eee sl cea as A4 48 4d 30 BA beadsnew z 20 C4 beadsold Fa ARA T EE E E E L MARA E CORNERO woe ASF SJ SA aos sacee 0 20 40 60 80 100 120 140 160 180 200 Adjusted MFI of 48 2 G1 Max 192 AUTO Sample Name list Figure 8 15
51. page 7 1 for more ormation Multi Graph view Figure 4 5 that displays different graphical for mats See The Multi Graph View on page 8 1 for more information There are two ways to show the Typing table To view the Typing table do either of the following Click the Typing table button under the file of interest in the Project manager Make a selection from the Window menu in the menu bar There are two ways to show the MultiGraph view To display the Multi Graph view do either of the following Click the Multi Graph button lil under the project of interest in the Project Manager Make a selection from the Window menu in the menu bar NOTE The graph view is blank until samples are selected for graphing To show or hide the Heat map click the Heat Map toolbar button MasterPlex GT www miraibio com 4 5 CHAPTER 4 GETTING STARTED Group locus names first row and allele names second row Typing Sample2 Sample2 gtp Bi fol gt 1 aie ell Name FA Total Events 5162 Sample Empty 48 1d 6305 Sample Empty E3 i es Fes Eee es ee ee po Beats gt te t24 fiese 16129 Andersor 16217 _ 16223T_ 16224 _ Andersor 16292T til i ai a ee ee Project Window toolbar 3 M beads only new 2253 3 z 5 B4 i 2 a B l l z E C4 ee a i 8 B2 47 1 5 4 153 65 135 2 BI aza 5519 JsampleEmetv 5 4 58 197 75 16
52. project gtp may be saved Displays the Typing table for the active results csv or gtp Displays the Multi Graph view for the active results Opens a window that displays the Homology table and chart for the active results Displays the Allele Call table for the Function gt Allele Call active results Opens the Report Manager Window gt Tiles the Typing table and Multi Cascade Graph views for the active results in a cascade Window gt Tile Tiles the Typing table and Multi Horizontally Graph views for the active results horizontally Window gt Tile Tiles the Typing table and Multi Vertically Graph views for the active results Debs eee vertically MasterPlex GT www miraibio com C 1 APPENDIX C TOOLBARS C 2 Typing Table Toolbar fo sja E0 Figure C 2 Typing table toolbar Table C 2 Typing table toolbar buttons and functions Typing Table Toolbar Button Function Opens the Parameter Setting dialog box for the active results csv or gtp Displays the relative intensity r of each allele in a group Displays the background adjusted MFI data for the alleles in the Typing table tess Displays the bead count data in the Typing B table Displavs the allele rows of the first locus group in the Typing table with a blue background Displavs the allele rows of the next locus with a white background This alternating use of color distinguishes the allele members of each group and
53. www miraibio com APPENDIX A APPLICATION OPTIONS Custom color field Luminosity slider Color swatch E Red e5 Sat 186 Green 234 ColorlSolid Lum 150 Blue 179 Cancel Add to Custom Colors Figure A 6 Color palette custom color options 4 To define a color use the click and drag operation to move the cross hairs in the custom color field Adjust the color brightness using the luminosity slider The Color swatch shows the color selection 5 When you are finished defining the color click Add to Custom Colors to apply the color and click OK The new color is displayed in the Applications Options dialog box 6 Click OK in the Applications Options dialog box to close the dia log box and apply the color to the Typing table gradient back ground 7 To apply the group colors to the Typing table choose the Use Allele Call Color for Gradation Background option Figure A 7 MasterPlex GT www miraibio com A 5 APPENDIX A APPLICATION OPTIONS Application Options Background Clustering Tool Plugins Bead Name Style Locus Name Allele Name Original Bead Name Start Up Window After Data Loading Show Table view C Show Graph View Table View Gradation Background Use Allele Call Color for gradation background Group Color 1 DJ Change Color Group Color 2 PE Change Color Heatmap Options Heatmap Bar Size 3 Pixel s Allele Reset All Cance
54. www miraibio com 4 11 CHAPTER 4 GETTING STARTED O sample Typing able dl Multi Graph sample2 Typing Table dl Multi Graph Figure 4 10 Project Manager Use a drag and drop operation to merge results 2 At the prompt click OK The Typing table displays the merged results The sample columns from the dragged file are added to the bottom of the Typing table and the well locations are numbered 2 A1 2 A2 2 A3 and so on If another file is merged the sample columns are added to the bottom of the table and the well locations are designated 3 A1 3 A2 3 A3 and so on Sample Merge Using the Batch Method 1 Inthe Project Manager right click the project of interest and select Merge All from the shortcut menu that appears Figure 4 11 gt Projects below the selected project with bead sets named identical to the bead set of the selected project are merged with the selected project 4 12 MasterPlex GT www miraibio com CHAPTER 4 GETTING STARTED C HLA A B DR BLD Projects below Sample2 TE Gane that use bead sets ill Multi Graph named identical to Di Samplez Sample2 bead sets are Typi view File Info merged with Sample2 dl Multi pelete ample Typin g Project Merge Wizard Typing Table Multi Graph Di SAmpleSmall Typing Table il Multi Graph Figure 4 11 Project Manager Sample Merge Using the Wizard 1 Inthe Project Manager right click the project of interest and select Proj
55. 09776 pg TE ae EG 47 2 Sort By Expression 150 140 163 143 111 71 c3 l472d Sort By Sample 216 192 204 192 135 80 BG 48 2 Cluster Analysis Genotype Expression 137 114 134 107 78 61 e2 l48 2 Reset 138 128 152 1201 83 60 c3 l48 2d 124 105 133 112 96 72 eE l47 4 Open Bar Graph 128 110 132 107 106 58 A2 148 4 Open Scatter Plat 148 170 212 1as 167 96 E2 a Local Negative Control ay Ht EM xa 45 a3 lt 1484 177 143 164 151 143 92 o Jara Ne isa tse 78 asaf tse Ag la8 4a 151 159 181 166 134 89 bi juza 7 fus Jsemoeemivi na na ws wif s as ab 7 E dS8L 77 ir8 tes 208 132 nes 99 ir Jes lees Jsampetmav 159 1500 a naf isr e heo jes sm SammieEmptv 150 133 nar asof nes 66 as ons 7 omal s ss KT gt Figure 7 9 Typing table Sorting by expression level MFI data 2 To view the homology score for a sample position the mouse pointer over the sample name A pop up tool tip displays the sample name and homology score Resetting the Sample Sort To reset the Typing table sample rows to the default the order in which the data were collected in the Luminex system do either of the following Click the Reset Sample Sort button Right click a sample row and click Reset Sample Sorting in the shortcut menu that appears The Typing table displays the sample rows in the order that the data were collected in the Luminex system 7 10 MasterPlex GT www miraibio com
56. 0T Anderson Anderson 16311C 16311C 16320 Anderson 16294T Anderson Anderson 16294T Anderson Anderson 16294T Anderson lt l C3 47 2d RI r G1 48 2 r G2 48 2 r G3 48 2d r E1 47 4 r a2 48 4 r E2 47 4 r a3 48 4 r E3 47 4d r A4 48 4d lt lt l l lt l lt l I lt l lt Sample name column Allele names displayed vertically Figure 7 10 Allele Call table Genotype call displayed vertically 2 Ifyou want to display multiple allele calls horizontally click the button gt The allele names are displayed side by side Figure 7 11 BI Allele Call Sample2 Sample2 gtp sajas m m XI Allele Call Allele Frequency Genotype Frequency Haplotype Frequency IA IB e F1 48 1 16126C Anderson C F2 48 1 16126C Anderson F3 48 1d 16126C Anderson A1 beads only new B4 beads new C4 beadsold B2 47 1 f B3 47 1d B1 47 1 C1 47 2 C C2 47 2 C3 47 2d G1 48 2 7 G2 48 2 G3 48 2d C E1 47 4 r A2 48 4 r E2 47 4 r a3 48 4 r E3 47 4d r a4 48 4d D1 47 3 D2 47 3 a uu Anderson Anderson Allele names Anderson Anderson Anderson IB I7E Anderson displaved Anderson Anderson CEIC 16311 16329 h o ri ZO nt al ly Anderson Anderson 16311C 16311C 16320T 16362T Anderson Anderson 16311C 16311C 163
57. 11C 16311C 16320T 16362T Anderson Anderson 16311C 16311C 16320T 16362T Anderson Anderson 16311C 16311C 16320T 16362T Anderson 16294T 4nderson 16362T Anderson 16294T Anderson 16362T Anderson 16294T 4nderson 16362T Anderson Anderson Anderson Anderson 16126C Anderson Anderson 962976 Anderson 16126C Anderson 16126C Anderson KIEKU IEEE did dds F Allele Call SampleSmall jam 38 Allele Call Allele Frequency Genotype Frequency Haplotype Frequency F E1 4 F D1 3 Figure B 13 Allele Call table Same genotypes painted top different genotypes painted bottom MasterPlex GT www miraibio com B 15 APPENDIX B PROJECT OPTIONS amp PARAMETERS Allele Call Table Options Enable reference sample Choose this option to display the reference selection sample selection radio buttons in the Allele Call table Figure B 14 Show when number Choose this option to help identify samples of alleles is fewer than the that have fewer called alleles than the reference reference sample Figure B 14 F Allele Call SampleSmall SampleSmall gtp Radio buttons for selecting a reference sample are available SES BI A BIE BIR Figure B 14 Allele call table reference sample selection enabled top disabled bottom B 16 MasterPlex GT www miraibio com APPENDIXB PROJECT OPTIONS a
58. 14 346 34 616 661 20 981 486 35 607 a 932 80 r8 xl Negative control Figure 5 1 Typing table The sample named no dna well A1 is a negative control Removing a Negative Control 1 Select the negative control sample row s The selected sample row s are highlighted 2 Right click a sample name in the highlighted selection and do either of the following If you are working with one results file click Local Negative Con trol in the shortcut menu that appears If you are working with merged files click Global Negative Control in the shortcut menu that appears The dash marks are replaced with the results data 5 3 Setting Negative Controls Automatically The MasterPlex GT software can automatically set the negative controls by identifying user specified key words in the sample name In the MasterPlex GT www miraibio com 5 3 CHAPTER 5 NEGATIVE CONTROLS Background tab of the Application dialog box you can set the keywords that identify a negative control 1 Select Option gt Set Application Options from the menu bar The Application Options dialog box opens Figure 5 2 IV Perform Automatic Background Sample Detection on data load Add New Keyword Background Keywords NOTE These parameters are effective only to new projects Reset All Cancel Figure 5 2 Application Options dialog box Background tab 2 Click the Background tab 3 Choose the option Perform Au
59. 163 0 42308 0 35350 0 0 32105 0 49405 0 42395 0 35439 O 0 32167 0 46246 0 45813 0 39433 0 32394 0 0 29340 0 49154 0 48381 0 41243 0 33920 0 30674 0 48291 0 47537 0 40571 0 33093 0 31525 0 29921 0 46800 0 49930 0 50343 0 46246 0 49154 0 48291 eaS 065208 0 65064 0 65860 0 46368 0 49163 0 49405 0 45813 0 48381 0 47537 em 0 67715 0 67667 0 68589 0 40198 0 42308 0 42395 0 39433 0 41243 0 40571 Mossos O NE 0 67401 0 67845 0 69039 0 33525 0 35350 0 35439 0 32394 0 33920 0 33093 0 65208 0 67715 0 67401 0 32237 0 33786 0 33874 0 30967 0 32233 0 31525 0 65064 0 67667 0 67345 MMMM 0 30469 0 32105 0 32167 0 29340 0 30674 0 29921 0 65860 0 68569 0 69039 0 99801 M 0 04798 0 05110 0 04988 0 04081 0 04075 0 03716 0 06143 0 05915 0 05343 0 06719 0 06337 0 06178 0 02620 0 02800 0 02531 0 02017 0 02071 0 01663 0 05219 0 05246 0 04792 0 03924 0 03606 0 03488 0 01477 0 01390 0 01324 0 01456 0 01303 0 01302 0 02707 0 02891 0 02920 0 03335 0 03421 0 03404 g gt Figure 7 22 Homology table 2 To copy the Homology table right click the table and select Copy Table As Text from the shortcut menu that appears 3 To view the Homology chart click the Homology Chart tab The window displays the Homology chart Figure 7 23 MasterPlex GT www miraibio com 7 23 CHAPTER 7 RESULTS TABLES Figure 7 23 Homology chart 4 To view information about a point in the chart position the mouse pointer over the point A pop up tool tip display
60. 2 48 3 F D2 47 3 D3 47 3d C D1 47 3 C C4 beadsold f E2 47 4 C E1 47 4 C A2 48 4 lt Anderson Anderson 16126C Anderson Anderson IB I7C Anderson 16126C Anderson 16126C Anderson KEE lt td lt lt lt lt lt lt lt aaqqaqa ARRESE lt lt l lt l Figure 7 17 Allele Call table sorted by expression Choose a reference sample top After the sort the reference sample appears in the stop row and allele calls are sorted in descending order of expression calls that differ from the reference are painted bottom MasterPlex GT www miraibio com 7 19 CHAPTER 7 RESULTS TABLES 2 To toggle the view and paint highlight the allele calls that are the same as the reference sample click the oa button gt Allele calls that are the same as the reference sample are painted with the group or allele color specified in the Parameter Setting dialog box see Group and Allele Color on page 6 8 Allele calls that are different from the reference sample are not painted Figure 7 18 amp Allele Call Sample2 Sample2 gtp sajas a m 8j xl Allele Call Allele Frequency Genotype Frequency Haplotype Frequency IA IB IC1 Ic2 ID IIA1 JIIAZ IIB Anderson Anderson 1631 1C 16311 163207 __ 16362T Anderson Anderson 16311C 16311C 163207 16362T Anderson 16311C 16311C 16320T 16362T 16294T Anderson 16362T 16294T Anderson 16362T 16294T Anderson 16362T
61. 2 INSTALLING MASTERPLEX GT The installation process begins and the InstallShield Wizard appears Figure 2 1 InstallShield Wizard E xl Welcome ta the InstallShield Wizard for HasterFlex GT The InstallShield Wizard mill install MasterPlex GT on your computer To continue click Next Cancel Figure 2 1 InstallShield Wizard 2 To continue the installation click Next The Choose Destination Location window appears Figure 2 2 InstallShield Wizard Choose Destination Location Select folder where Setup will install files Setup will install MasterPlex GT in the following folder Ta install ta this folder click Mest To install to a different folder click Browse and select another folder Destination Folder C Program FileskHitachi5ott MasterFles GT Browse Vrs ee Cancel Figure 2 2 Install Shield Wizard Choose Destination Location 2 2 MasterPlex GT www miraibio com CHAPTER 2 INSTALLING MASTERPLEX GT 3 Confirm the default destination folder or click Browse to specify a different folder The destination folder is where the program files will be installed 4 Click Next The program is installed When the installation is complete the InstallShield Wizard Complete window appears Figure 2 3 InstallShield Wizard InstallShield Wizard Complete Setup has finished installing MasterPlex GT on your computer 4 Back Figure 2 3 InstallShield Wizard Co
62. 20T 16362T Anderson 16294T Anderson 16362T Anderson 16294T Anderson 16362T Anderson 16294T Anderson 16362T IN U d SI SI SI XI SI SI SI SI SI SI SI SI SI Figure 7 11 Allele Call table Genotype call displayed horizontally 7 12 MasterPlex GT www miraibio com CHAPTER 7 RESULTS TABLES 3 To copy the current view of the Allele Call table right click the table and select Copy Table as Text or Copy Table as Text With out Quotations from the shortcut menu that appears The table information is copied to the system clipboard 4 To print the current view of the Allele Call table right click the table and select Print Allele Call Table from the shortcut menu that appears 5 Click the Close button to close the Allele Call table Merging Wells Sometimes an assay format stipulates the same sample in several different wells and probes each well with a different bead set In the Allele Call table you can merge the different wells and view the complete genotype results across all wells for the sample Figure 7 12 MasterPlex GT www miraibio com 7 13 CHAPTER 7 RESULTS TABLES FI Allele Call Sample2 Sample2 gtp Allele Call Allele Frequency Genotype Frequency Haplotype Frequency IA IB IA1 IIA2 IIB e F1 48 1 Y 16126C Anderson f F2 48 1 16126C Anderson F3 48 1d 16126C Anderson A1 beads only new f B4 beads new C4 beadsold f
63. 23T 0 00 16224C 0 00 Anderson 92 31 16292T 16295T 0 00 16294T 50 00 16294T 16296T 0 00 16294T 16296T 16304C 0 00 16298C 0 00 16304C 0 00 Anderson 50 00 16309G 0 00 16311C Figure 7 19 Allele Call table Allele Frequency tab Genotype Frequency The Genotype Frequency tab Figure 7 20 displays the frequency and percentage for each allele or combination of alleles called at each locus group 1 To view the genotype frequency information click the Genotype Frequency tab 2 To copy the genotype frequency information right click the table and click Copy Table as Text from the shortcut menu that appears The table information is copied to the system clipboard MasterPlex GT www miraibio com 7 21 CHAPTER 7 RESULTS TABLES FI Allele Call Sample2 Sample2 gtp sajas m m j XI Allele Call Allele Frequency Genotype Frequency Haplotype Frequency Genotype 50 00 91 67 8 33 50 00 50 00 50 00 50 00 100 00 50 00 50 00 50 00 50 00 50 00 50 00 100 00 100 00 ow vownwunrwownunwoe w Figure 7 20 Allele Call table Genotype Frequency tab Haplotype Frequency The haplotype frequency tab Figure 7 21 displays the frequency and percentage for the genotypes that were called 1 To view the haplotype frequency information click the Haplotype Frequency tab 2 To copy t
64. 25 0 of total intensity Intensity Threshold 135 MFI C Intensity based Allele Call Call anything bigger than so MFI as an Allele Apply to all groups loci MFI Allele Ratio Figure 3 9 Parameter Setting dialog box Allele calling parameters and options MasterPlex GT www miraibio com 3 11 CHAPTER 3 BEFORE YOU BEGIN FI MasterPlex GT Typing SampleSmall SampleSmall gtp MEA File Edit View Function Option Window Help ax Do a laul Sale aall Locus SNP1 SNP2 SNP3 SNP O Sample2 Beats ft mt et mt tit wt mt f Typing Table WellName SamoeName _ TotalEvents notes TT L 3 il Multi Graph G G FTI MINNI AME 21 576 40 D SampleBase Typing Table ill Multi Graph D SampleSmall Typing Table ill Multi Graph MFI Adjusted MFI Count FEV 151 2 30 7 SNPImt 50 69 13 8 649 85 0 131 234 286 0 122 2 922 380 3 41 2 SNP3mt 109 648 59 3 693 166 5 24 0 MM Project Manager Tvping table in the Project Window includes file tree top and statistics table bottom Figure 3 10 Project Manager and Project Window Tvping table displaved in the Project Window 3 12 MasterPlex GT www miraibio com CHAPTER 3 BEFORE YOU BEGIN BI MasterPlex GT Graph SampleSmall me hal Fie Edit View Option Window Help ex Dolea ele Hull Slaa aall VJ D SampleSmall kal F m ju 3 mi R Al IE E A EEEEEETEEEEEET CEL vao Typi
65. 311C Sample V Delete Project for Merge 16311C 16320T Samplel 16319A Sample Cancel 16320T Samplet Anderson Samnist Figure 4 19 Laver Merge wizard LaverSampleA Locus A Bead Name Locus A MV Suffix All Beads for this Project Figure 4 20 Edit bead name dialog box The box title is the selected project LayerSampleA and bead name Locus2 A MasterPlex GT www miraibio com 4 19 CHA PTER 4 GETTING STARTED 4 20 To edit the allele name choose the Allele option and enter an allele name To edit the suffix name choose the Suffix option and enter a suffix name To apply the new allele and suffix names to all loci in the project choose the All Beads for this Project option Click OK MasterPlex GT www miraibio com CHAPTER NEGATIVE CONTROLS This chapter explains negative controls and how to set them A negative control NC can be set manually in the Typing table or the Multi Graph view The MasterPlex GT software can also automatically identify negative controls based on keyword recognition 5 1 Local and Global Negative Controls The MasterPlex GT software computes a background value the average MFI of the negative controls and subtracts it from the sample bead set MFI to obtain the background adjusted sample MFI data A local negative control is specific to a particular set of result A global negative control is applied to merged results Both local and global negative contr
66. 5 307 0 c1 2 1 2 75 47 71 203 5 87200000 808 JSampleEmetv mid ERA o a c3 2 2 2 63 45 67 195 5 G1 3 1 1 60 44 60 187 5 82002020 lsi35 JsampleEmetv Le a ae Bae tas aa 63 1 1 2 48 38 51 191 3 E1 4740000000 faoss Sample Empty o 2 1 1 1 1 1 1 E2 o o 2 17 1 o 1 1 1 A3 o 1 1 2 3 1 2 2 2 E3 47 4d 6462 Sample Emoty 2 1 2 1 2 a 1 2 2 Ad 1 1 2 2 2 o 1 2 1 D1 1 2 a 3 3 1 2 2 4 D3 47 3d 6952 Sample Empty 2 2 4 3 3 2 Hi 6188 Sample Empty 2 2 1 3 2 1 4 3 3 4 Well Sample Totalbeads Notes entered in the location name sample counted per Luminex software Figure 4 4 Project Window Typing table displaying MFI data Multi Graph toolbar Pos 1 Sample Name Al beads only new Ci 472 E1 474 FI 484 G1 48 2 H1 48 3 Well 4 H B2 474 on D2 47 3 E2 474 G2 48 2 H2 48 3 a 48 0 47 1d 47 2d 47 3d 47 4d 48 1d 48 2d 48 3d 48 4d beads new beadsold Sample name D3 E3 F3 G3 H3 Ad B4 C4 Peterlee Det at het tacit Pod tet beg ETT Fol Vitis oe Onl Poet teat at Lomt fet et es ted Dek yg U Tey CTT GUN hav eat deed OTT Heat map Figure 4 5 Project Window oE ml J SJ s f B mao van L TE Muti Compare Ty Depth EE Threshold Editing E Sample by Sample E Allele by Alle Adjusted MFI of 47 1 B1 IA 16124C 16126C 16129A Anderson 18 i 162231 MFI Background la B ict io D iat a2 IB ic iD Adjusted MFI of 47 3 D1 Ta 3 4007 lis j
67. 6 ji 285 fi 273 ji 271 117 35 116 33 M 125 3 JIM 128771 a 3 2 1 1 000 m331c 000 m44 3a 000 000 2 000 ren 2000 2000 m 7 000 000 000 tame tay op taa c a e en i Blue color gradient represents Red color gradient represents relative intensitv for alleles relative intensitv for alleles called at the first locus 92R7 called at the next locus Amel Figure A 4 Typing table gradient background The default gradient background alternating red and blue shows relative expression levels of the alleles called in each sample MasterPlex GT www miraibio com A 3 APPENDIX A APPLICATION OPTIONS NOTE When you change the Group 1 or 2 color the new color is also applied to the graph points in the Sample by Sample scatter graph and the x axis and y axis thresholds in the Allele by Allele scatter graph Ii To select a different Group 1 or Group 2 color 1 Inthe Applications Options dialog box Figure A 1 click Change Color for Group 1 or Group 2 The color palette appears Figure A 5 Custom colors EE HH EE Ea Define Custom Colors gt gt Figure A 5 Color palette 2 To select a predefined color click one of the basic colors 3 To define a custom color click Define Custom Colors The color palette shows the custom color options Figure A 6 A 4 MasterPlex GT
68. 6320T lt lt lt r C1 47 2 KU r C2 47 2 C3 47 2d xl C G1 48 2 G2 48 2 G3 48 2d C E1 47 4 A2 48 4 E2 47 4 f 43 48 4 E3 47 4d lt m Anderson Anderson Anderson Anderson Anderson Anderson a a FF A m a heed T PUE a amp a FI 3 8 EZ 2 m KI lt lt l l lt lt l lt l lt I lt l BG Z KEON x Allele Call Allele Frequency Genotype Frequency Haplotype Frequency e F1 48 1 16126C Anderson r F2 48 1 16126C Anderson C F3 48 1d 16126C Anderson r B4 beads new C4 beadsold f B2 47 1 B3 47 1d f B1 47 1 C1 47 2 C2 47 2 C3 47 2d C G1 48 2 f G2 48 2 G3 48 2d f E1 47 4 A2 48 4 f E2 47 4 r A3 48 4 E3 47 4d C A4 48 4d r D1 47 3 D2 47 3 Anderson Anderson Anderson Anderson Anderson 629 6 Anderson Anderson Anderson 16311C 16311C 16320T 16362T Anderson Anderson 16311C 16311C 16320T 16362T Anderson Anderson 16311C 16311C 16320T Anderson 16294T Anderson Anderson 16294T Anderson 16294T Anderson ltd lt td lt 1 lt 0 lt 1 lt 1 lt lt a lt I lt I x gt T A e 2 2 a ILI IL IL IL IL ILL Figure B 12 Allele Call table Vertical allele name list top horizontal allele name list bottom MasterPlex GT www miraibio com B 13 APPENDIX B PROJECT OPTIONS amp PARAM
69. 7 5 F a 1 a H8 97121 as 1 0 7 50 2 0 1 1 o HS 2 3536 2 1 9 2 ct 1 3 2 c7 gt z 5 J4 E7 mesom 2 feo d O 3 18 2 8 4 45 0009 118 o 9 0 zi cB 10 15 2 12 7 Bi 10 105 1 1 0 9 E8 mesmo eooo d O 15 1 7 3 as 88 2 1 1 1 a c9 9 6 a 3 1 2a 2 61 1 1 0 0 bg 0 18 3 64 oft 51 1 0 0 0 Ag 53 8 4 z i EA 42 0 3 1 1 F9 55 12 3 1 0 ES aa o 1 0 9 G7 1 20 5 3 5 sz a7 1 9 1 o E9 10 27 3 1 5 ssf 5 82 0 1 1 1 B5 al 2 2 9 2 a 13 3 9 2 0 c5 A 3 9 si si a 13 2 9 1 1 D5 me2369 eooo 2 Bi sl l i 76 1 14 2 1 0 G5 h16236 12 6000 Sample Empty 2 a 2 mil 2 ae 2 14 2 0 1 ly Alleles columns of Alleles of Alleles of the locus IC2 have blue the next next locus IIB background locus ID have blue have white background background Figure 7 2 Typing table Stripe background view uses alternating color blue or white to identify the alleles columns associated with a locus You can select different data views in the Typing table Relative intensity Displays the percent relative intensity Figure 7 4 page 7 5 for the alleles at a locus Median fluorescence intensity Displays the background adjusted MFI Figure 7 5 page 7 6 MFI of the alleles Bead count Displays the number of bead events Figure 7 6 page 7 7 counted for sample acquisition MasterPlex GT www miraibio com 7 3 CHAPTER 7 RESULTS TABLES You can view the Typing table with a striped Figure 7 2 or a gradient backgr
70. 920 0 33093 0 65208 0 67715 0 67401 0 32237 0 33786 0 33874 0 30967 0 32233 0 31525 0 65064 0 67667 0 67945 0 30469 0 32105 0 32167 029340 0 30674 0 29921 0 65860 0 685869 0 69039 SSE NOSSEOT INN 0 04798 0 05110 0 04988 0 04081 0 04075 0 03716 0 06143 0 05915 0 05343 0 06719 0 06337 0 06178 0 02620 0 02800 0 02531 0 02017 0 02071 O 01663 0 05219 0 05246 0 04792 0 03924 0 03606 0 03489 0 01477 0 01390 0 01324 0 01456 0 01303 0 01302 0 02707 0 02891 0 02920 0 03335 0 03421 0 03404 w gt Figure 3 14 Homology table Correlation coefficient between sample genotypes are displayed 3 14 MasterPlex GT www miraibio com CHAPTER 3 BEFORE YOU BEGIN Figure 3 15 Homology chart The correlation coefficients between sample genotypes are plotted MasterPlex GT www miraibio com 3 15 CHAPTER 3 BEFORE YOU BEGIN TE Multi Compare Th Depth EE Threshold Editing EE Sample by Sample EE Allele by Allele Adjusted MFI of 47 1 B1 lA 161240 161260 161294 OS Anderson 1B mo 16217 162237 1622740 mW Anderson ICi 162927 16295T D 162947 1 H mm mm Dl mm mm moraa e E 200 100 MFI Background Sees J J 1 1 1 LI LI 1 1 1 Ur a kata Abate tit lli ID I amp i 42 IB li IID Adjusted MFI of 48 3 H1 ELE LIA i 16124C toes alee OO A e sani pao testa a 161260 ge ii GAME GA A 181208 a be k d i ee Snderson 500 T E is
71. A 3 Typing table Original Bead Name option selected Start Up Window After Data Loading The Project Window can display the Typing table or Multi Graph view when you open a results file csv or a project gtp Choose Show Table View to display the Typing table when the Project Window opens A 2 MasterPlex GT www miraibio com APPENDIX A APPLICATION OPTIONS Show Graph View to display the Multi Graph view when the Project Window opens Table View Gradation Background The Typing table with the gradient background Figure A 4 uses a color gradient to indicate the relative expression level of the alleles called at each locus in a sample a lighter shade represents a lower expression level Alternating colors defaults are blue and red distinguish the alleles rows of adjacent loci groups in the table For example in Figure A 4 the alleles of the first locus 92R7 are highlighted with a blue color gradient that represents the relative expression levels of the called alleles a lighter color shade indicates a lower expression level In the next locus Amel a red color gradient Locus group names first row allele names next row Locus 92R7 Amel DYS199 J Beats c eS eS as ae E 35 DYS391 a M9 e le WellName JSample Name Total Events notes sl cm55 2a 2000 2000 273 271 f 285 ji 236 ji 294 i 269 ff 291 fi 297 ji 301 fi 291 fi 289 ji 291 fi 265 l 283 ji 27
72. A version h 4 Group Name Reset Task e Type Standard Beads Cross Talk General Figure 10 33 Lookup Table Editor 5 Click Export The Export Wizard appears Figure 10 34 Export Wizard Check All Uncheck All Entry Name HLA Lookup Table Name of the lookup table for export cut Figure 10 34 Export Wizard 6 To export all of the loci information click Check All Alternatively select individual items for export 7 Click Export The Save As dialog box appears Figure 10 35 10 26 MasterPlex GT www miraibio com CHAPTER 10 GENOTYPING USING A LOOKUP TABLE Save in B SampleData fa e E File name HLA Lookup T able att Save as type Lookup Table att x Cancel Figure 10 35 Save As dialog box 8 Confirm the destination directory and file name and click Save The lookup table gtt is saved to the selected directory MasterPlex GT www miraibio com 10 27 CHAPTER 10 GENOTYPING USING A LOOKUP TABLE Copying a Lookup Table The currently displayed contents of a lookup table can be copied to a tab delimited text file 1 Right click the lookup table and select Copy as Text from the shortcut menu that appears The contents of the current view of the lookup table are copied to the system clipboard 2 Paste the clipboard contents to the application of interest for example Notepad Deleting a Lookup Table 1 Open the Lookup Tab
73. AAALLLLILALALLALAALLLILASALLLLALLLLLLLAALLL LILLIA Figure 3 20 Dendrogram Example cluster analysis results MasterPlex GT www miraibio com 3 19 CHAPTER 3 BEFORE YOU BEGIN 3 20 MasterPlex GT www miraibio com CHAPTER GETTING STARTED Working with Luminex Results This chapter explains how to open Luminex results csv It also explains how to combine or merge results Results can be merged two different ways a sample merge combines results across experiments that use identically named bead sets a layer merge combines the results from different bead sets that probe the same sample 4 1 The Project Manager and Project Window The Project Manager and Project Window appear when you open Luminex results csv In MasterPlex GT the results are called projects and include the the Typing table graphs or dendrogram created in the Multi Graph view parameter settings user selected samples A project can be saved gtp for future sessions Project Manager Displays a Tile tree of open results projects and Figure 4 1 the Statistics table Project Window Displays the Typing table or Multi Graph view of Figure 4 2 the results data The Project Manager is anchored or docked to the Project Window Figure 4 3 You can undock the Project Manager from the Project Window This enables you to change the position of the Project Manager relative to the Project Window More than one project may be open at a time and each
74. Allele Call Allele Frequency Genotype Frequency Haplotype Frequency IIA1 IIA2 IIB f F1 48 1 IV 16126C Anderson F2 48 1 V 16126C Anderson V 16126C Anderson A1 beads only new f B4 beads new C4 beadsold f B2 47 1 Anderson Anderson f B3 47 1d Anderson Anderson f B1 47 1 Anderson 62176 Anderson CENAE Anderson Anderson 16311C 16311C 163207 163627 Anderson 16311C 16311C 163207 16362T luster Analysis Genotype Expression Anderson 16311C 16311C 16320T 16362T Copy Table As Text 16294T Anderson 16362T Copy Table As Text Without Quotations 16294T Anderson 16362T Print Allele Call Table 162941 Anderson Heil C E3 47 4d C A4 48 4d KUKUK 7 IL IL IL IL IL ILI Reference sample top row for the sort amp Allele Call Sample2 Samplz2 gtp alge m xl Allele Call Allele Frequengf Genotype Frequency Haplotype Frequency IA IB IC1 IC2 ID IA1 IIA2 IIB Anderson Anderson 16311C 16311C 16320T 163627 Anderson Anderson 16311C 16311C 16320T 16362T Anderson Anderson 16311C 16311C 16320T 16362T Anderson 16294T Anderson 16362T Anderson 16294T Anderson 16362T Anderson 16294T Anderson 16362T Anderson Anderson C1 47 2 r C2 47 2 C3 47 2d f G1 48 2 G2 48 2 f G3 48 2d f B2 47 1 f B3 47 1d F3 48 1d f B1 47 1 f Fi 48 1 F2 48 1 A1 beads only new H1 48 3 f H3 48 3d H
75. CHAPTER 7 RESULTS TABLES 7 4 Allele Call Table The Allele Call table displays the alleles called for each sample Figure 7 10 In the Allele Call table you can view genotype or haplotype sort samples by homology to a user selected reference sample view allele frequency locus group frequency or genotype fre quency 1 To view the Allele Call table open the project of interest and do one of the following click the iat button right click the Typing table and click Allele Call in the shortcut menu that appears select Function Allele Call from the menu bar A separate window opens and displays the Allele Call table Figure 7 10 The alleles are highlighted using the group or allele color specified in the Parameter Setting dialog box see Group and Allele Color on page 6 8 MasterPlex GT www miraibio com 7 11 CHAPTER 7 RESULTS TABLES BI Allele Call Sample2 Sample2 gtp po S w m XI Allele Call Allele Frequency Genotype Frequency Haplotype Frequency IA IB IC1 IAI JHAZ JIIB Allele ie F1148 1 V 16126C Anderson F2 48 1 IV 16126C Anderson names C F3 48 1d IV 16126C Anderson f A1 beads only new C B4 beads new C4 beadsold f B2 47 1 Anderson Anderson B3 47 1d Anderson Anderson f B1 47 1 Anderson 62076 Anderson r C1 47 2 Anderson Anderson 16311C 16362T 16311C 163207 Sample C1 C c2 47 2 Anderson Anderson 16311 haplotype 16311 1632
76. E 34 22 0 133 185 7 96 3 216 248 8 115 1 144 164 0 114 1 228 257 9 113 2 24 22 4 91 8 247 279 5 113 4 67 69 5 103 6 51 20 2 39 3 IM9G 100 77 4 77 2 144 154 1 107 2 37 30 3 91 4 7 64 98 5 80 91 9 114 7 286 326 3 114 0 55 58 6 107 0 141 150 1 106 5 g File tree Statistics table Figure 8 1 Project Manager MasterPlex GT www miraibio com 8 1 CHAPTER 8 GRAPHS In the Multi Graph view Figure 8 2 the sample list displays the samples in the active results The project manager displays MFI and count data for samples that are highlighted in the sample list The Multi Graph view displays each type of graph in a separate tab Table 8 1 provides a brief summary of each graph type DIRE Pos Sample Name l JE Multi Compare ae Depth E Threshold Editing E Sample by Allele t_4 gt m bm33 1a a bmd4 3c Adjusted MFI of bm33 1a B1 ART S re bm44 3b a m C MIT C1 bm33 1b E Amel D1 bm331c 5 400 x Hi bm44 3a 2 wim32 5b Fi se l ors l 200 a a wim32 5 z C3 cm39 So 100 eo l A A3 cm396a a A m G H2 wim32 5c 92R7 Amel la DL ja 3225 Tat w SEA ee Adj d MFI of wim32 5a F2 S D2 cm37 4b juste ene Saa 92R7 mc C2 cmar 4a 500 i E2 cm374c B Amel Bi cm55 2c 8 400 X 293 Flt cm55 2a 300 D5199 je Cae Alten iL er D3 ren 100 c 452 0 G 92R7 Amel DYS391 SRY 465 SRY 3225 Tat MB Adjusted MFI of cm55 2b F1 S kud 92R7
77. ETERS Allele Call Table Background Coloring You can sort the Allele Call table by expression level MFI data or haplotype to a user selected reference sample The Allele call table displays the reference sample in the top row of the table Paint alleles with color if genotype is same as the reference Paint alleles with color if genotype is different from the reference Choose this option to paint highlight alleles in the Allele Call table that have the same genotype as the user selected reference sample Figure B 13 The group or allele color is applied depending on what was specified in the Parameter Setting dialog box Choose this option to color alleles in the Allele Call table that is do not have the same genotype as the user selected reference sample Figure B 13 The group or allele color is applied depending on what was specified in the Parameter Setting dialog box B 14 MasterPlex GT www miraibio com APPENDIXB PROJECT OPTIONS amp PARAMETERS BI Allele Call Sample2 Sample2 gtp sales jwa xl Allele Call Allele Frequency Genotype Frequency Haplotype Frequency C1 47 2 C2 47 2 C3 47 2d F G1 48 2 G2 48 2 G3 48 24 r B2 47 1 F B3 47 1d r F3 48 1d F F1 48 1 F F2 48 1 F H1 48 3 F H3 48 3d M H2 48 3 F D2 47 3 Fr D3 47 3d F D1 47 3 C4 beadsold F E2 47 4 F E1 47 4 IF A2 48 4 am Anderson Anderson 163
78. G A LOOKUP TABLE New Type Name Entry Type Mame A a01 omo Figure 10 13 New Type Name Entry dialog box 5 Enter a type genotype name for the selected locus or blood type and click OK The Lookup Table Editor displays the new type genotype name Figure 10 12 oF NOTE The short name is not used in MasterPlex GT 2 0 EN To edit a tvpe name select the tvpe name and click the Edit Tvpe Name button El Alternatively right click the Lookup Table Edi tor and select Edit Type Name from the shortcut menu that appears The Edit Type Name Entry dialog box appears Figure 10 14 Edit Type Name Entry Edit Type Mame Figure 10 14 Edit Type Name Entry dialog box 7 To delete a type name select the type name and click the Delete button T Alternatively right click the Lookup Table Editor and select Delete from the shortcut menu that appears Click Yes in the confirmation message box that appears 8 To specify the display color of the type name in the Typing table a Double click the color swatch The color palette appears Figure 10 15 10 12 MasterPlex GT www miraibio com CHAPTER 10 GENOTYPING USING A LOOKUP TABLE Basic colors mi mm Be ATE ee LE A Ue mean EEE eee BEE eee Custom colors eee TTT Define Custom Colors gt gt Figure 10 15 Color palette b To select a predefined color click one of the basic colors c To define a custom color click Define Cus
79. IB mi Doe eee bt Taare ga AEE re ea ene D 200 9344 gt a 16223T Ba ia ifa agir ae Iai a fanal root RR aa een 162246 100 i i i h l f H Anderson MAL ART KANA A a dil LA IB ic Ic2 ID 144 HAZ IB 162947 Adjusted MFI of 47 3 D1 161240 pesasse POSea cate isin a pie ES i 161260 E iii i 16129A B l l sess IAEA i Anderson pee _ fii 8 D eee eee AEDE EEEE sae ell 200 i ns i 16223T eae PA i Te l 162240 100 i j l Anderson l 1 l l U L i r ICI 1 G 162927 16295T IA IE Z1 Ice D Ad WAS IE 16294T Figure 3 16 Multi Compare bar graphs The graphs displav allele MFI or RI values for user selected samples 3 16 MasterPlex GT www miraibio com CHAPTER 3 BEFORE YOU BEGIN Gamplesmall Timiiiin sire Ia l i t riain a a i jaq b II ge Nae aaa el a aia eee tee ini it l aan a Ia ae a ara p A a Pes ie oe a m s a o o oOo a Oo Oo GG dm 6 6 6 c oo D im qT co T H gpunoJE yeg 4 SMP1 SMP2 SNPS SMPA SMPS SMPE SNP SNPS SMFS SNPIO Figure 3 17 Depth bar graph Each bar graph shows MFI or RI values for user selected samples www miraibio com 3 17 MasterPlex GT BEFORE YOU BEGIN Figure 3 18 Sample by Sample scatter graph Each point represents an allele Figure 3 19 Allele by Allele scatter graph Each point represents a sample 3 18 MasterPlex GT www miraibio com CHAPTER 3 BEFORE YOU BEGIN Pig ALLAL AALA L
80. ICENSE You may a Use the Software on a single machine at any given time b Obtain limited numbers of Copy Protection Devices Additional Copy Protection Devices are provided only as a convenience of running the software c In no manner engineer or reverse engineer the copy protection hardware or whole or part of the software d Copy the software only for backup provided that you reproduce all copyright and other proprietary notices that are on the original copy of the Software provided to you Certain Software however may include mechanisms to limit or inhibit copying Such Software is marked copy protected e Transfer of the Software and all rights under this Agreement to another party together with a copy of this Agreement if the other party MasterPlex GT www miraibio com i LICENSE AGREEMENT agrees to accept the terms and conditions of this Agreement If you transfer the Software you must at the same time either transfer all copies whether in printed or machine readable form to the same party or destroy and copies not transferred RESTRICTIONS You may not use copy modify or transfer the Software or any copy in whole or in part except as expressly provided for in this Agreement Any attempt to transfer any of the rights duties or obligations hereunder except as expressly provided for in this Agreement is void YOU MAY NOT RENT LEASE LOAN RESELL FOR PROFIT OR DISTRIBUTE TERM This Agreement is effectiv
81. LAIM BY ANY OTHER PARTY SOME STATES DO NOT ALLOW THE LIMITATION OR EXCLUSION OR LIABILITY FOR INCIDENTAL OR CONSEQUENTIAL DAMAGES TO THE ABOVE LIMITATION OR EXCLUSION MAY NOT APPLY TO YOU MasterPlex GT www miraibio com lil LICENSE AGREEMENT GOVERNMENT LICENSEE RESTRICTED RIGHTS LEGEND If you are acquiring the Software on behalf of any unit or agency of the United States Government the following provisions apply The Government acknowledges Mirai s representation that the Software and its documentation were developed at private expense and no part of them is in the public domain The Government acknowledges Mirai s representation that the Software is Restricted Computer Software as that term is defined in Clause 52 227 19 a of the Federal Acquisition Regulations FAR The Government acknowledges that the Software is classified as Commercial Computer Software and the Government is acquiring only restricted rights in the Software and its documentation will be as defined in Clause 52 227 19 c 1 and 2 of the FAR Manufacturer is MiraiBio Inc 1201 Harbor Bay Parkway Suite 150 Alameda CA 94502 EXPORT LAW ASSURANCES You acknowledge and agree that the Software is subject to restrictions and controls imposed by the United States Export Administration Act The Act and the regulations thereunder You agree and certify that neither the Software nor any direct product thereof is being or will be acquired shipped t
82. LOOKUP TABLE WI Lookup Table Selection Entry Mame Last Modified Edit Selected Entry PROJECT Create Mew Lookup Entry HLA Lookup Table 10 31 2003 12 56 50 PM HL4 4 HLAB HLA DR Blood Type Verdi 10 VYer1 10 Veri l0 verl 00 Delete Selected Entry Select Latest Tables Unselect All OK Cancel Figure 10 8 Lookup Table Selection window This window shows the available lookup tables and the table applied to the current project In this window you can create a new lookup table or edit a table 4 Inthe Lookup Table Selection window Figure 10 8 click Create New Lookup Entry The New Lookup Table Entry dialog box appears Figure 10 9 New Lookup Table Entry Lookup table name HLA Lookup Table carcer Figure 10 9 New Lookup Table Entry dialog box 5 Enter a name for the new lookup table and click OK The Lookup Table Editor appears Figure 10 10 10 8 MasterPlex GT www miraibio com CHAPTER 10 GENOTYPING USING A LOOKUP TABLE SI Lookup Table Editor Export Apply Reset All M 4 O8 Oor O ao Locus Setting Name HLA A Version 1 1 Group Name Reset ao aoz aos aos aos Ja stdija stdz Click a task button to change the view in the Lookup Table Editor Figure 10 10 Lookup Table Editor Type view Lookup Table Funcion Editor Button Export Opens the Export wizard Apply Applies the changes to the selected lookup table and close the Lo
83. RA Aa od on ina ab ied ta jint i len oat aa aa eaves ea au ea sbraa aa lie ba fist ma Sa las bats sa aa oft A Relative Intensity wn o SNPI NP2 SNP3 SNP4 SNPS SNP6 SNP SNPS SNPS SNP10 Relative intensity threshold 25 Figure 8 14 Threshold Editing tab Relative intensity data NOTE The allele is called in the sample if the intensity data exceed the MFI threshold and the relative intensity threshold Changing the Intensity Thresholds You can change the MFI or relative intensity threshold for individual alleles 1 Position the mouse pointer over the threshold segment that you want to change gt The mouse pointer changes toa F 2 Use the drag and drop method to move the threshold to a higher or lower intensity The allele calls are updated using the new threshold The new intensity threshold is displayed in the Parameter Settings dialog 8 18 MasterPlex GT www miraibio com 8 7 CHAPTER 8 GRAPHS box For more information see Parameter Settings and Options on page 6 1 Sample by Sample Scatter Graph The Sample by Sample scatter graph plots the allele bead set MFI data for two user selected samples The graph displays the correlation coefficient value R for the two samples and distinguishes between alleles that are called in both samples only the x axis sample only the y axis sample or neither sample dy Open the Multi Graph view for the results you want to graph and click the
84. SING A LOOKUP TABLE FI MasterPlex GT Typing HLA A B DR BLD 0 File Edit View Function Option Window Help Disma ole r aa O HLA A B DR BLD Typing Table il IT Graph Ss es SES ee ee ee ee E i fe tt wWellNeame Samole Name TotaiEvents notes oof l Fae Pe ese a si jawi fees e aooo lasso __ bt jaws T 1 fiaoooa agro l foun suis 1 lot fiago0os C m eooor Jeo KI Acjusted MFI Count a2 faos fes O Sead Tae so ev G e R e ca fomo fons aE b2 fwon e o e2 foma fsa F2 fona fse ooo ae e Figure 10 38 MasterPlex GT application window 3 Click the Parameter Setting button bh The Parameter Setting dialog box appears Figure 10 39 Parameter Setting Group set are gt Cancel Save setting as Import Setting T Parameter setup for the individual bead Minimum Events po o count for each bead IV Use group color for Chart and Allele Call Table Lookup Table Lookup Table Allele Name Report Intensity Call Inten A Other HLA A v1 10 Other HLA B v1 10 Group Allele Identifier Group Prefix of beads in this group 7 Edit Bead Names Group Name Ja IG Change Color Ploidy Diploid C Haploid Other Apply this Ploidy to all groups loci eu 7 ja Apply to all alleles in the Allele Name NIE CONS same order in each group M Apply to all same name alleles r Allele Call Parameters for A
85. SNP10 Figure 8 8 Depth graph Relative intensity data for samples 1 7 8 5 Multi Compare and Depth Graph Display Options Several options are available for the Multi Compare or Depth graph display see Table 8 2 on page 11 You can also modify the graph view in the following ways change the y axis scale increase the graph width or height show or hide allele name tags reposition the allele name tags modify the bottom graph axis labels magnify a user selected area of the graph move the graph inside the Project Window 8 10 MasterPlex GT www miraibio com CHAPTER 8 Table 8 2 Graph display options Default 3 dimensional 3D graph Show name tags for called alleles Display the graph legend Display graph bars using solid colors Paint only the called alleles Display MFI data Display the group name of the bead horizontally on the bottom axis of the graph Display only the group name of the bead on the bottom axis of the graph Display all labels on the bottom axis of the graph To Toggle the view between 3D and 2D view Hide or unhide the name tags Hide or unhide the legend Toggle the view between a solid or gradient color graph bar Toggle between paint all alleles or paint only the called alleles Display relative intensity data RI Return the display to MFI data Display the group names vertically on the bottom axis of the graph Display the group name and the allele
86. Sample by Sample tab Click the amp button and click the Sample by Sample tab The Multi Graph view is now in the wo sample comparison mode ill NOTE To plot a scatter graph the Multi Graph view must be in the two sample comparison mode In the Sample Name list Figure 8 15 right click one of the samples you want to plot in the scatter graph and select Sort By Expression from the shortcut menu that appears The selected sample is moved to the top of the sample list and the remaining samples are sorted by similar expression level MFI data descending order In the sample list click the second y axis sample for the scatter plot The Sample by Sample scatter plot is displayed Figure 8 15 The graph points are identified by color Graph Point Color Represents an allele that is White Not called in either sample Red default Called only in the y axis sample Blue default Called only in the x axis sample Black Called in both samples MasterPlex GT www miraibio com 8 19 CHAPTER 8 GRAPHS NOTE You can change the red and blue default colors for alleles in the Sample by Sample scatter graph in the Application Options dialog box See Changing the Gradient Background Colors on page A 3 Hi 5 To view an allele name tag put the mouse pointer over a graph point A pop up tool tip displays the allele name 6 To display all of the allele name tags click the graph All
87. Tables Unrzelect All OK cancel Figure 10 11 Lookup Table Editor Type view top Lookup Table Selection window bottom 10 10 MasterPlex GT www miraibio com CHAPTER 10 GENOTYPING USING A LOOKUP TABLE 1 Click a tab to select the 2 To specify a color for the type name displayed locus Click a task icon to in the Typing table double click the color change the Lookup Table swatch to open the color palette Editor view BI Lookup Table Editor Export Apply Reset All Locus Setting Name Version 10 Group Name Reset 3 Enter the genotype 4 Click to select the alleles frequency that you want to include expression on in the expression pattern Figure 10 12 Lookup Table Editor Type view The Type icon and locus tab icon change from yellow to blue YD to indicate the type has been edited Lookup Table Editor MI Description Component Type Name Genotype name Short Name Not used in MasterPlex GT 2 0 Frequency Genotype expression frequency If a type has ambiguity candidates this value determines the order of a call and its ambiguity candidates in the Typing table Frequency must be gt 0 4 Click the Add New button EE Alternatively right click the Lookup Table Editor and select Add New from the shortcut menu that appears The New Type Name Entry dialog box appears Figure 10 13 MasterPlex GT www miraibio com 10 11 CHAPTER 10 GENOTYPING USIN
88. Thresholds tab of the Multi Graph view For more information see Threshold Editing on page 8 16 6 4 Intensity Based Allele Call For this method the MasterPlex GT software calls an allele if the MFI is greater than a user specified value m Click the 8 button to display the Parameter Setting dialog box Figure 6 2 2 Ifyou want to set parameters for one particular allele bead type only select the allele name in the upper box and choose the Parameter setup for the individual bead option NOTE If this option is not chosen the parameter settings are applied to alleles on a per group basis Confirm the default or enter a new Minimum Events value Choose the Intensity Based Allele Call option Confirm the default or enter a new MFI threshold value Click Apply The parameter settings are applied to the active results a y MasterPlex GT www miraibio com 6 5 CHAPTER 6 ALLELE CALL PARAMETERS 6 5 Editing a Bead Name You can edit the bead name For more information about prefix group and allele names see Bead Name Conventions on page 3 1 1 Click the amp button to display the Parameter Setting dialog box Figure 6 5 2 Click Edit Bead Names The Edit Bead Names dialog box opens Figure 6 4 Edit Bead Names Figure 6 4 Edit Bead Names dialog box 3 Select the bead name you want to edit and enter a new name 4 Click OK when you finish editing the names The new bead name
89. Typing table 2 Click Edit Bead Name in the pop up menu that appears Figure 3 6 The Change Bead Name dialog box appears Figure 3 7 3 Edit the bead name and click OK 3 6 MasterPlex GT www miraibio com CHAPTER 3 BEFORE YOU BEGIN FI MasterPlex GT Typing Sample2 Sample2 gtp File Edit View Function Option Window Help Dieta SEJ al Safes Blo Be de ahei Well Name Total Events Sample Empty Sample Empty bt Tbeeds new La7er Tsar Encty ca beadsold os Sample Emety Bo fart st Same Emoty is2 7 jsis ai nets taa jus umeta eo Of Of NORANAI ln OO amp WNHNHNH AD HH I I aN BO BH S amp S wO SE Figure 3 6 Typing table Right click a bead name to edit the name Change Bead Name New Bead Name Figure 3 7 Change Bead Name dialog box Editing the Group or Allele Name Only You can also edit just the group or allele name in the Parameter Settings dialog box 1 Inthe Parameter Settings dialog box select the group or allele name you want to edit The Group Name or Allele Name box below displays the selected name component Figure 3 8 For example the allele name 16217C is selected in Figure 3 8 MasterPlex GT www miraibio com 3 7 CHAPTER 3 BEFORE YOU BEGIN al Parameter Setting Group set Cancel Save setting as Import Setting iv Parameter setup for the individual bead Minimum Events ko count for each bead IV Use group color for Chart and Al
90. USER MANUAL Version 2 0 for Microsoft Windows MasterPlex GT 2 0 Genotype Analysis Software MiraiBio A HITACHI SOFTWARE COMPANY For Research Use Only 1201 Harbor Bay Parkway Suite 150 Alameda CA 94502 TELEPHONE 1 800 624 6176 1 510 337 2000 FACSIMILE 1 510 337 2099 Part no P 33120 10250 TRADEMARKS MicroSoft is a registered trademark of Microsoft Corporation Luminex is a registered trademark of Luminex Corporation COPYRIGHT 2002 2003 MiraiBio Inc All Rights Reserved LICENSE AGREEMENT LICENSE AGREEMENT BEFORE OPENING THIS PACKAGE YOU SHOULD CAREFULLY READ THE FOLLOWING TERMS AND CONDITIONS BY OPENING THIS PACKAGE YOU AGREE TO BECOME BOUND BY THE TERMS AND CONDITIONS OF THIS AGREEMENT WHICH INCLUDES THE SOFTWARE LICENSE AND LIMITED WARRANTY IF YOU DO NOT AGREE WITH THESE TERMS AND CONDITIONS YOU SHOULD PROMPTLY RETURN THE PACKAGE UNOPENED TO MIRAIBIO INC Mirai or Mirai Distributor AND YOUR MONEY WILL BE REFUNDED The enclosed software is licensed not sold to you for use only upon the terms of this Agreement and Mirai reserves any rights not expressly granted to you You are responsible for the selection of the Software to achieve your intended results and for the installation use and results obtained from the Software You own the media on which the Software is originally or subsequently recorded or fixed but Mirai retains ownership of all copies of the Software itself L
91. Vv Use group color for Chart and Allele Call Table PAR Table Lookup Table Tallele Name Report intensity Call inten Other HLA amp v1 10 Other HLA B v1 10 s i Group Allele Identifier Group Pretix of beads in this group 7 Edit Bead Names Group Name ja fe Change Color Ploidy Diploid C Haploid Other Apply this Ploidy to all groups loci Allele Name r Allele Call Parameters for A C Use Relative Intensity for Allele call Reportable Level 50 of total intensity Intensity Threshold MFI Intensity based Allele Call Call anything bigger than so MFI as an Allele Apply to all groups loci Figure 10 4 Parameter Setting dialog box c Click Lookup Table The Lookup Table Selection window appears Figure 10 8 d Confirm that the Lookup table Selection window includes the name of the table Entry Name that you imported 10 4 MasterPlex GT www miraibio com CHAPTER 10 GENOTYPING USING A LOOKUP TABLE BI Lookup Table Selection Sel Entry Mame Last Modified d Selected Gries PROJECTS Create Mew Lookup Entry HLA Lookup Table 10 31 2003 12 56 50 PM HL44 HL46 HLADF Blood Type Verdi 10 VYer1 10 Yert 10 verl 00 Delete Selected Entry Select Latest Tables Unselect All Figure 10 5 Lookup Table Selection window This window shows the lookup tables installed in the MasterPlex GT software Component in the Lookup Function Table Selection Window
92. a n a 900 800 700 ee ee oes ne 600 Adjusted MFI of 9 B2 500 Sett tal a eae Seta at bl jaw eae ee eae ees AEE aco i 300 0 200 400 600 800 1 000 1 200 1 400 Adjusted MFI of 1 B1 Figure 7 26 Sample scatter graph Multi Graph view in Two Sample Mode only the top sample in the Sample Name list and one other user selected sample can be displayed in the Multi Graph view To exit this mode when you are done viewing the Sample by Sample scatter graph click the ad button a NOTE Opening the Sample by Sample scatter graph puts the MasterPlex GT www miraibio com 7 27 CHAPTER 7 RESULTS TABLES 7 28 MasterPlex GT 3 www miraibio com CHAPTER GRAPHS The MasterPlex GT software can plot the MFI data in the following graphical formats Multi Compare bar graph Depth bar graph Sample by Sample scatter plot Allele by Allele scatter plot Heat map This chapter explains how to work in the Multi Graph view 8 1 The Multi Graph View To display the Multi Graph view for the active results click the lill button a particular project in the Project Manager click Multi Graph under the file of interest in the file tree Figure 8 1 O Sample Typing Table ill Multi Graph D Sample2 Typing Table il Multi Graph D SampleBase Typing Table ol eT D SampleSmall Tying Tan ill Multi Graph MFI Adjusted MFI Count Bead Ave sb cz ame B
93. a o o2 o2 o2 o5 Stdi 05 Stdi Std2 Stdi Std2 Std2 05 Std2 Std2 E1 ido004 7 Figure 10 43 Allele Call window MasterPlex GT www miraibio com 10 33 CHAPTER 10 GENOTYPING USING A LOOKUP TABLE 10 5 Ambiguity Candidates Sometimes there is more than one possible genotype for a sample This occurs when the allele MFIs that exceed threshold match more than one expression pattern in the lookup table The Typing table shows the number of ambiguity candidates for a sample in the Am column Ifa sample has ambiguity candidates the Type column shows the genotype with the highest frequency e To view the ambiguity candidates for a genotype double click the number in the Am column Figure 10 44 Double click the number again to collapse the list of ambiguity candidates 8 Jo mji gt u Bil Jie A e or e 5200 pi laoo sees awan Ne A A soo pe j l p dua D1 lioo fara Jaoor oo E ts a ka ha Z esan JET E1 laoo 5 Os aoo Es 21 e1 fioo 52 fos aoo of 17 Ho fiaoooz faa6o ff on amo ol a2 figooos_ Masas fn aoo Es 21 Ba Jisooos ss Jo Jaoozaoos of a c2 figooto cots faos En D2 laoo Jas Jaoos ora of 17 E2 laom f stp faos aor of ssi F2 laona Jssa8 oo or ofie e2 liona 5o35 hoos aoo Ea 1s H2 lions less3 aoo aoo a Type for sample A i Double click the Am id0001 A 003 A 010 number to display the Am
94. aan cae cuore Sis004 a FEE or sea 100 0 ri pren ME m A ir f A IB ict C2 IDIAIAZIB Adjusted MFI of 47 2 C2 ae i i i 5 400 ee EU p fet 163207 de L Seen eee jee amp 300 C BoT Ta ee fi 200 ajo oO 400 0 A B ict 2 D wat a2 B e 1D Adjusted MFI of 48 1 F2 TA 161240 2 16126C 88 16129A 0 Anderson 12 18 16217C 142 16223T 61 182240 132 WL mae OME 907 LILL YM z ra m Ima ima In ae ma Graph legend Multi Graph view shows the Multi Compare graphs for the selected samples 4 6 MasterPlex GT www miraibio com CHAPTER 4 GETTING STARTED Viewing Project Information 1 To view project information right click the project name in the Project Manager and select View File Info from the shortcut menu The Project information is displayed Figure 4 6 Project Information D samplet lt lt C Program FilesiHitachiSoft MasterPlex GT SampleData SampleBase csv gt gt Typing Table Program LUMINEX Build VERSION 1 7 69 ill Multi Graph Date 5 29 02 1 23 45 PM lik Sample2 Serial Number Lx10000000000 Typi Tabi Session 0000 00 00001 yping lable Operator SAMPLE il Multi Graph Heater Temparature 55 D SampleBa Number Of Samples 25 U ET Minimum Events 20 view File Info Typing T dl Multi Gre Delete O SampleSme Typing T P i g Merge All dl Multi Gre Project Merge Wizard Figure 4 6 File information 2 To copy the file information to the
95. all Inten A Other HLA A v1 10 Other HLA B v1 10 v E MGroupiAllele Identifier Group Prefix of beads in this group 7 Edit Bead Names Group Name Ja ma Change Color Ploidy Diploid C Haploid Other Apply this Ploidy to all groups loci ms API to all alleles in the Allele Name Change Color same order in each group ia jA to all same name alleles Allele Call Parameters for A C Use Relative Intensity for Allele call Reportable Level 50 of total intensity Intensity Threshold P MFD e Intensity based Allele Call Call anything bigger than fo MFI as an Allele Apply to all groups loci Figure 10 31 Parameter Setting dialog box 3 Click Lookup Table The Lookup Table Selection window appears Figure 10 32 BI Lookup Table Selection Entry Mame Last Modified PROJECT HLA Lookup Table 10 31 2003 12 56 50 PM HL44 HL46 HLADF Blood Type Verdi 10 VYer1 10 Yert 10 verl 00 Figure 10 32 Lookup Table Selection window Edit Selected Entry F Create Mew Lookup Entry Delete Selected Entry Select Latest Tables Unselect All Cancel MasterPlex GT www miraibio com 10 25 CHAPTER 10 GENOTYPING USING A LOOKUP TABLE 4 Click the table that you want to export and click Edit Selected Entry The Lookup Table Editor appears Figure 10 33 FI Lookup Table Editor Apply Reset Alll O 4 8 OG or O er Locus Setting Name HLA
96. am vo Pos 1 Sample Name al TEE Multi Compare Th Depth EE Threshold Editing Sample by Sample E Allele by Allele Al nodna C1 SampleSmall Nm GI r womonHn non w MFI Background SNP2 SNPS SNP4 SNM5 SNPS SNP SNPB SNPS SNP10 Sample list Intensity threshold MFI 35 Allele that was not called in Called allele the selected sample Figure 8 13 Threshold Editing tab MFI data 2 To view a pop up tool tip that displays the sample name and inten sity data position the mouse pointer over a graph point MasterPlex GT www miraibio com 8 17 CHAPTER 8 GRAPHS ejjejal ml fy Blea afer si ME el ano vao Pos l Sample Name idl TEE Multi Compare Th Depth EE Threshold Editing Sample by Sample E Allele by Allele Al nodna lye SampleSmall 100 Claire ET14 95 I ee ee i eu al Deer ee cecal SIN e bos seooe lean an Seah ae one ne serene TIR PR G1 6 OD RL ne Rare Ay VOPR es ERT PRR CE ny SR ER COTES T SIRT ORES GRRE SE EP Hi 7 A2 8 DD AJ B2 9 75 B3 10 70 65 b mnijla es aa tezi tale sal esi set aq a ea tfa a ea E Sa 98 Ba en Sat aa mal saroli aa ea saved Ga tenna di bala ep Satan 59 eaten qali ee sa ea ins 02 tea bl sated ent ot am aa putea ana jon sa tea aoi Sa b8 ea sa a ie sal be Sal Sa ma sara ea IA Ia 9 60 ka sala toll edios taa ont pal Sa sa ale aa a aa sam ea eu oa liv ba lan aj eur eaten ea Geral a as oa en coated rea as ell oni ba ERRE
97. an be saved as a project gtp A project includes the results file s csv in the Typing table graphs or dendrogram created in the Multi Graph view parameter settings user selected samples 1 Click the Save button ei Alternatively select File gt Save Project from the menu bar The Save As dialog box appears Figure 6 14 6 14 MasterPlex GT www miraibio com CHAPTER 6 ALLELE CALL PARAMETERS Save in SampleData ek Fil MJHLA A B DR BLD gtp is Sample gtpi ju Sample2 gtp u SampleBase gtp ju SampleSmall gtp File name Sample1_2 atp Save as type MasterPlex GT Project File gtp x Cancel Figure 6 14 Save As dialog box 2 Confirm the default or enter a new location where you want to save the file 3 Enter a file name for the project gtp and click Save Using the Save As Function Use the Save As function if you change the project for example change a parameter setting or option and want to save it without overwriting the previously saved file gtp 1 Select File gt Save Project As from the menu bar The Save As dialog box appear Figure 6 14 2 Confirm the default or enter a new location where you want to save the project 3 Enter a file name for the project gtp and click Save Opening a Project You can use the menu bar toolbar or a drag and drop method to open a project gtp Using the Menu Bar or Toolbar 1 To open a previously saved proje
98. ard for the same locus follow step 3 to step 8 11 To delete a standard click the standard row and click the Delete button T Alternatively right click the Lookup Table Editor and select Delete from the shortcut menu that appears Click Yes in the delete confirmation message that appears 12 To specify standards at another locus follow step 1 to step 10 MasterPlex GT www miraibio com 10 17 CHAPTER 10 GENOTYPING USING A LOOKUP TABLE Specifying Cross talk In the Cross talk view of the Lookup Table Editor Figure 10 17 you can specify the per cent cross hybridization between two bead sets The software uses this information to compute MFI values that are corrected for cross hybridization For example if bead sets A B and C exhibit cross hybridization as follows 10 of allele B binds to bead A 5 of allele C binds to bead A then the software computes Corrected MFI Original MFI 0 1 MFI 0 05 MFI 1 Inthe Lookup Table Editor click a tab to select a locus 2 Click the Cross talk button Cl The Cross talk view for the selected locus or blood tvpe appears Figure 10 23 el Lookup Table Editor Export Apply Reset All O4 O8 O or Oe Locus Setting Name HLA A Version f 4 Group Name Reset Task DJ A 01 aoz JAQD3 ao aos JA Stdi a std Type Standard Beads Figure 10 23 Lookup Table Editor Cross talk view In this view you can specify the per
99. are graph and Depth graph Ploidy Choose a ploidy option for the sample data Note Other ploidy is the same as haploid The type of ploidy affects the allele frequency calculation Allele Call Parameters Use Relative Intensity The software calls the allele if all of the following for Allele Call conditions are met Rl tiele 2 user specified RI threshold MF Litcie 2 user specified intensity thresh old Intensity Based Allele The software calls the allele if the MF yj gt Call user specified absolute intensity threshold B 2 MasterPlex GT 3 www miraibio com APPENDIXB PROJECT OPTIONS amp PARAMETERS Parameter Setting Group set gt Cancel Save setting as Import Setting V Parameter setup for the individual bead Minimum Events 20 count for each bead NV Use group color for Chart and Allele Call Table Lookup Table arete eroupname Type Lookup Table Allele Name Reportable Level Intensity Threshold Call Intensity 161 26C 161294 Anderson 16217C 16223T 16224C Anderson 25 0 16292T 16 25 0 16294T 25 0 b Group Allele Identifier peel Group Prefix 1 of beads in this group 4 tt Edit Bead Names Group Name fia Change Color Ploidy Diploid Haploid Other Apply this Ploidv to all groups loci Apply to all alleles in the Allele Name fi 6124C Change Color same order in each group Apply to all same name alleles
100. beads onlvnew 2253 Sample Emoty Ba beadsnew zraz Sample Emoty C4 beadsold a51 JSampieEmetw a m JI KT sa es ias Da 5 6 3 158 74 140 300 wa ssi Semoleemoiv 4 5 4 MT 7s tes a07 0 2 1 21 75 a7 np 203 2 2 2 2 74 39 e9 isa 47 2d Sample Empty 1 2 2 2 63 45 67 70185 o 3 1 60 aa eo 187 a2 fas semoene 1 4 e o so as se o 1 1 2 48 38 51 MAI 4740000000 s0e3 JSampeEmetv 1 0 2 1 1 1 1 2 1 1 21 1 1 2 2 0 0 2 1 1 0 1 1 0 1 1 21 3 1 2 2 2 1 2 1 2 0 1 2 1 1 2 2 2 0 1 2 47 3 1 2 1 sl 3 1 2 2 1 1 2 1 a 3 5 3 473d 952ml Emoty 0 2 2 4 3 3 1 6188 2 2 1 3 2 1 4 3 A 1 4 7 a A a 2 Figure B 5 Typing table gradient background B 3 Graph View The settings in the Graph View tab Figure B 6 specify the defaults for the Multi Compare and Depth graph display FI New Project Default Parameters Allele Call General Options i Show Legend IV Show Sample labels T aqs Label Tilt 0 to 30 degree Allele Bar Graph Options WW View 3D Graph M Show Gradation IV Paint Called Allele Only IV Graph MFI Value Use Relative Intensity if not checked Show MFI RI threshold lines in bar graphs Bottom Axis Labels Vertical Label F Thin out labels Show Locus name and Allele Name Heatmap Options IV Show Heatmap OK Reset All Cancel Figure B 6 New Project Default Parameters Graph View tab B 6 Mas
101. between sample genotypes A bar graph of the background adjusted MFI or RI values for a user selected sample A composite bar graph of the background adjusted MFI or RI values for user selected samples A scatter plot of the background adjusted MFI data for a user selected pair of samples Each graph point represents an allele A scatter plot of the background adjusted MFI data for a user selected pair of alleles Each graph point represents a sample A diagram of the sample cluster analvsis results www miraibio com CHAPTER 3 BEFORE YOU BEGIN a Parameter Setting Group set Raines Cancel Save setting as ll Import Setting T Parameter setup for the individual bead Minimum Events po 20 count for each bead F Use group color for Chart and Allele Call Table Lookup Table Lookup Table Allele Name Report kalb Call Inten A SNP1 SNP1 Diploid 25 0 SNP2 _ SNP2 Diploid 25 0 35 SNP3 SNP3 Diploid 25 0 35 SNP4 SNP4 Diploid 25 0 35 i j lt E Group Allele Identifier Group Prefix SMF of beads in this group 2 st Edit Bead Names Group Name Er Change Color Ploidy Diploid Haploid Other Apply this Ploidy to all groups loci r s Apply to all alleles in the Allele Name Change Color same order in each group Apply to all same name alleles Allele Call Parameters for SNPI1 Use Relative Intensity for Allele call Reportable Level
102. biguity ambiguity candidate s candidate Figure 10 44 Typing table 113 SS Se ae a ji Po Beats PIU Jon foo Jos Jos stot sta Wet Nene _fSanle Nene__ fetal Evers potas eA Ane _F__f ___ ____j__j_____ At blank 5200S Ng beers 3 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 The Am column shows the number of ambiguity candidates for a type Double click the Am number to view the ambiguity candidates for a particular type 10 34 MasterPlex GT www miraibio com 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 CHAPTER 10 GENOTYPING USING A LOOKUP TABLE 10 6 Inversion Candidates An inversion candidate is a genotype that is a possible call if the expression level of one allele in the expression pattern is changed to its opposite for example an allele that is expressed MFI gt threshold is changed to not expressed MFI lt threshold The Typing table shows the inversion candidates for a sample in the Iv column Figure 10 46 Checking the inversion candidates is useful way to check for errors especially for rare combinations of alleles or other possible genotypes when a no match is called To view the inversion candidates for a genotype 1 Click the Gradient Background button __ Results that exceed the MFI threshold are highlighted blue in the A locus tab red in the B locus tab and alternating r
103. cent cross hybridization between two bead sets 3 Click the Add New button EE Alternativelv right click the Lookup Table Editor and select Add New from the shortcut menu that appears The New Standard Bead Name Entry dialog box appears Figure 10 19 10 18 MasterPlexGT 3 www miraibio com CHAPTER 10 GENOTYPING USING A LOOKUP TABLE New TargetBead Entry TargetBead Name o Figure 10 24 New Target Bead Entry dialog box 4 Click the drop down arrow and make a selection from the drop down list and click OK The MFI of the selected allele will be corrected for cross hybridiza tion 5 The bead set is added to the Lookup Table Editor Figure 10 25 ge Lookup Table Editor Export Apply Reset All 4 0 8 Oor oO ew Locus Setting Name version oo Group Name Reset Task J IT DJ ji aoz Jaos Jaos aos fasta astd2 0 o Type et 0 0 0 0 Standard Beads Cross Talk General Figure 10 25 Lookup Table Editor Cross talk view 6 Enter the per cent cross hybridization data for the allele pairs MasterPlex GT www miraibio com 10 19 CHAPTER 10 GENOTYPING USING A LOOKUP TABLE x Lookup Table Editor Sele Export Apply Reset All 5 4 O8 Oor Oe Locus Setting Name HLA A version 11 1 Group Name Reset Task E DJ ao lao lao lace laos Jasta JA std Type 4 Di 0 03 0 0 02 0 0 Standard Beads Cross Talk Ge
104. ch allele in a group For example in a biallelic analysis of allele a and allele b RL MFI MFI MFI x 100 RI MFI MFI MFI x 100 The software calls an allele if the following conditions are met Rl ticle gt a user specified RI threshold reportable level MF Lee 2 a user specified intensity threshold 1 Click the button to display the Parameter Setting dialog box Figure 6 2 MasterPlex GT www miraibio com 6 3 CHAPTER 6 ALLELE CALL PARAMETERS em Parameter Setting SEE Group set Cancel OK Save setting as Import Setting Parameter setup for the individual bead Minimum Events 20 count for each bead Use group color for Chart and Allele Call Table Lookup Table Prefix GroupName Type Lookup Table Allele Name Reportable Level Intensity Threshold Call Intensity A 35 92R7 92R7 Other Amel Amel Other 35 DYS1 DYS199 Other 35 DYS3 DYS391 Other 35 M9 M9 Other 35 s Group Allele Identifier Group Prefix of beads in this group 2 Edit Bead Names Group Name 92R7 Plaidy Diploid Haploid Other Apply this Ploidy to all groups loci Allele Name m r Allele Call Parameters for 92R7 e Use Relative Intensity for Allele call Reportable Level 25 0 94 of total intensity Intensity Threshold 35 MFI C Intensity based Allele Call Call anything bigger than MFI as an Allele Apply to all groups loci MFI Allele Ratio Figur
105. click Lookup Table Click the By Locus button ES and Show Type button The Typing table displays a tab for each locus and the blood type and a column of genotypes for the selected locus or blood type Figure 10 41 MasterPlex GT www miraibio com 10 31 CHAPTER 10 GENOTYPING USING A LOOKUP TABLE Click a tab to view results for a locus or blood type Type column aye DT 2 2u e pem Am Ja jor e essenenenanenenane oe ome eo oo a 7 doz Jos 1 WellName Sample Name __ TotalEvents Notes Tvperay FF Jam iv ff d At blank O No Matches oj 9 5200 No Matches o a Jaooor 566 Jaos amo e CA so so ooo oon ET TA 77 R T E C E E B E bt e jesa E ss taste E 4000 m b ea o aosan A o a a tal VC E fs ea Te a I r eer ee eef rof a ier faooog ssa IT aco 0 7 tes ee 50S at 1000 1800 i jemor seo a oe cro jozojiiej 181 7101 ter sa 57710001 100 sa jam CS mil sl aa al l E cago fwar toa 0 t000 1000 e k a O aea a 111 118 111 18 8 7 10001 100 a Se a aC E E il aa el a a a aano S o ef nef nef mal el mal jez jiswa fos Jason acne Baf isf eo nso ae 200 266 wooo ono 124 33 124 113 120 1000 1000 Figure 10 41 Typing table Type Column Indicates Background Color Red No matching genotype found Yellow Automatic genotype call Blue Type or Am column Ambiguity candidate or number of ambiguity candidates Green Type or Iv column
106. ct gtp click the Open Project File button ta Alternatively select File gt Open Project File from the menu bar The Open dialog box appears Figure 6 15 MasterPlex GT www miraibio com 6 15 CHAPTER 6 ALLELE CALL PARAMETERS Open Look in SampleData tsi HLA A_B_DR_BLD gtp Sample1 gtp Sample2 gtp SampleBase gtp SampleSmall gtp Files of type MasterPlex GT Project File atp xl Cancel Figure 6 15 Open dialog box Project files gtp 2 Double click the project file gtp you want to open The project window for the selected file opens Using the Drag and Drop Method 1 Navigate to the project file gtp you want to open 2 Use the drag and drop operation to place the file in the Master Plex GT application window Figure 6 16 The Project Manager and Project Window appear Figure 6 16 In the Project Manager the file tree displays the file name The Project Window displays the Typing table default ii NOTE For more information about the Project Manager and Project window see page 4 1 6 16 MasterPlex GT www miraibio com CHAPTER 6 ALLELE CALL PARAMETERS E MasterPlex GT File Edit View Option Window Help Disimjaj ee Hull Soleo aln it SampleData File Edit View Favorites Tools Help GTT File 7KB Microsoft Excel Comma Separated Values File 46KB GTP File ke 35KB Microsoft Excel Comma Separat
107. ct MiraiBio Technical support at MiraiBio Inc 1201 Harbor Bay Parkway Suite 150 Alameda CA 94502 USA Tel 1 510 337 2000 Toll Free 1 800 624 6176 Fax 1 510 337 2099 E mail gene miraibio com Wwww miraibio com 1 2 MasterPlex GT 3 www miraibio com CHAPTER INSTALLING MASTERPLEX GT This chapter explains the minimum hardware and software requirements needed to install and use MasterPlex GT It provides installation instructions for a computer connected to the Luminex system 2 1 Requirements For optimum performance MasterPlex GT requires hardware and software that meet or exceed the following specifications It is also strongly recommended that you use the Luminex XY platform Hardware Requirements Platform IBM PC compatible Memory RAM 64 MB RAM or higher for Windowse 98SE 128MB or higher for Windows 2000 XP Storage space HDD 20 MB available space for the installation Inout devices Keyboard and mouse or any other pointing device Video RAM AMB or higher Monitor resolution SVGA 1024x768 pixels or higher Monitor color 16 bit color high color or higher CD ROM drive Required tor CD media version Not applica ble for download version Software Requirements Operating system Microsoft Windows 98SE 2000 XP only 2 2 Installing MasterPlex GT 1 Insert the MasterPlex GT CD ROM in the workstation computer and double click MasterPlex GT exe MasterPlex GT www miraibio com 2 1 CHAPTER
108. ct drop down list Figure 4 17 The wizard shows all open projects that can be merged with the selected project each sample has the same well location across all experiments By default all of the projects are selected for the merge 4 Confirm the projects selected for the merge or remove the check mark from a project that you do not want to include in the merge To remove all check marks right click the wizard and select Uncheck All from the shortcut menu that appears To check mark all of the projects right click the wizard and select Check All in the shortcut menu MasterPlex GT www miraibio com 4 17 CHAPTER 4 GETTING STARTED project the Project Merge wizard displays the name in red If this occurs the merge cannot proceed For information on how to edit a bead name see Editing a Bead Name on page 4 19 oF NOTE If a project includes a bead name that is used in another r Choose the Delete Project for Merge option to remove all but the top project name from the Project Manager after the merge For example in Figure 4 18 only the project LayerSampleA will be displayed in the Project Manager after the merge O LayerSampleA Project Manager Typing Table shows all open dl Multi Graph projects D LayerSampleC Typing Table dl Multi Graph O LayerSampleG Typing Table il Multi Graph O LayerSampleT Typing Table dl Multi Graph SI Layer Merge Wizard Top Project Locus Beads Project arid ke
109. e 6 2 Parameters Setting dialog box 2 Ifyou want to set parameters for one particular allele bead type only select the allele name in the upper box and choose the option Parameter setup for the individual bead NOTE If this option is not chosen the parameter settings are a applied to alleles on a per group basis 3 Confirm the default or enter a new Minimum Events value 4 Choose the Use Relative Intensity for Allele Call option 5 Confirm the default or enter a new Reportable Level When the analysis is biallelic the Parameter Setting dialog box also displays a bar graph of the MFI allele ratio specified by the Sader level For example the parameter settings in Figure 6 3 ca l an allele if MFI MFI MFI x 100 gt 25 6 4 MasterPlex GT www miraibio com CHAPTER 6 ALLELE CALL PARAMETERS 25 33 333 33 333 100 100 Allele Call Parameters for 92A 7 f Use Relative Intensity for Allele call Reportable Level 125 0 of ttal intensity Intensity Threshold C Intensity based Allele Call Call anything bigger than 50 MFI as an Allele as Allele Rati ele Ratio Figure 6 3 MFI allele ratio MFI allele ratio shows the minimum biallele ratio required to meet the reportable level 6 Confirm the default or enter a new Intensity Threshold MFI 7 Click Apply The parameter settings are applied to the active results NOTE You can also set the relative intensity thresholds in the
110. e until terminated You may terminate it at any time by destroying the Software together with all copies in any form This Agreement will immediately and automatically terminate without notice if you fail to comply with any term or condition of this Agreement You agree upon termination to promptly destroy the Software together with all copies in any form LIMITED WARRANTY Mirai warrants for the period of ninety 90 days from the date of delivery of the Software to you as evidenced by a copy of your receipt that 1 The Software unless modified by you will perform the function described in the documentation provided by Mirai Your sole remedy under the warranty is that Mirai will undertake to correct within a reasonable period of time any marked Software Error failure of the Software to perform the functions described in the documentation Mirai does not warrant that the Software will meet your requirements that operation of the Software will be uninterrupted or error free or that all Software Errors will be corrected 2 The media on which the Software is furnished will be free from defects in materials and workmanship under normal use Mirai will at its option replace or refund the purchase price of the media at no charge to you provided you return the faulty media with proof of purchase to li MasterPlex GT www miraibio com LICENSE AGREEMENT Mirai Mirai will not have any responsibility to replace or refund the purchase p
111. ea L A Mb ae nsiet ae 8 A iu iat eae ee ee aE ea so 6 1004j kesa ittie aaa Kiri ee ae pene jem E f WAW 24 PA AT JA IA IB Ic1 Ic2 ID 1A1 IA2 IIB lc Adjusted MFI of 48 3d H3 z So 8 Se eS ee E S 300 i Beene L pee See TE ne wee 736 Fa E F 195C ae 200 koa Dan D EEANN A ETERA rpa bA araa IS ene yaa 100 i i l J a aay 2a Seem aoa 2 Ad to WT t A A B set Ic2 ID WA1 A2 IB Ic e kg AA bill ax 340 AUTO v axis maximum al JEF Multi Compare Th Depth EE Threshold Editing EE Sample by Sample EE Allele by Allele je Adjusted MFI of 47 1 B1 IA IB Ic1 Ic2 ID Ad WA2 IIB lic ID Adjusted MFI of 48 3d H3 oe Ay js oat aed een pa 8 80 SOOO jiniiis Sai air N eee LE Coenen ie Oars a Ili B 820 H Zz HART A t IA IB ica 1c2 ID wat IA2 IB i iisttinta m ae y IA i i 100 i ri eae i i 16124 5 i 8 so erie ees ESL petri ose frat tars emia tae eaten 7S 16126C 5 IM Beton cares NE EEE EE ee eee E EEEE IAEA u 16129A 3 H 3 60 ei ee ee rep eet eee eee eee here Anderson 58 1 78 40 u e i LIB il n aS Pn yee merae ano n a e r mm 16217C 197 ji o ammi e a a BA 4a I Ic1 Ic2 ID HA1 142 IIB IC ID I E Adjusted MFI of 48 2 G1 3 Bio WE A 1 eed ee 4 Ss i oh eta e C PARE i H Jj cent Baer f U Il Jnana B 4 4 jet Il e IMMA IM Wa B MB 5
112. ect Merge Wizard from the shortcut menu that appears Figure 4 12 The Project Merge Wizard appears Figure 4 13 X Di HLA A B DR BLD Typing Table ill Multi Graph k Typing iew File Info ill Multi Delete LI Sample2 Typing al Multi t Merge All C SampleB Project Merge Wizard Typing Table al Multi Graph LI SampleSmall Typing Table dil Multi Graph Figure 4 12 Project Manager MasterPlex GT www miraibio com 4 13 CHAPTER 4 GETTING STARTED Project Merge Wizard Sample Merge Merge Some projects with the same bead set Layer Merge p Merge Some projects with the same Wells Figure 4 13 Project Merge Wizard 2 Click the Sample Merge button The wizard displays a drop down list of projects that are open in the Project Manager Figure 4 14 Sample Merge Wizard Top Proj l Lee Drop down list of projects in the Project Manager Make a selection from the list vi Samplet_2 of open projects v Sample1_3 v Sample1_4 List of projects that can be sample merged with the project selected above If you do not want to include a project in the merge remove the check mark Accept Cancel Figure 4 14 Sample Merge Wizard 3 Make a selection from the Top Project drop down list The wizard shows all projects that can be sample merged with the selected project results that have identically named bead sets By default all of the projects are selected for the merge 4 14 Mast
113. ected 2 To change a group color choose the option Use group color for Chart and Allele Call Table To change an allele color do not choose this option 3 Click the group name or allele name with the color that you want to change The Group Name color swatch or Allele Name color swatch shows the selected color Figure 6 7 and Figure 6 8 MasterPlex GT www miraibio com 6 9 CHAPTER 6 ALLELE CALL PARAMETERS Parameter Setting Group set Cancel OK Save setting as Import Setting Parameter setup for the individual bead Minimum Events 20 count for each bead Use group color for Chart and Allele Call Table Lookup Table Prefix Group Name Type Lookup Table Allele Name Reportable Level Intensity Threshold Call Intensity SNPI i 35 35 36 wt allele in group SNP4 selected SNPS Diploid j i Group Allele Identifier Group Prefix of beads in this group 2 Edit Bead Names Group Name Ploidy GF Dipl F Haploid ve roups oci Apply to all alleles in the Allele Name wt TV Change Color same order in each group Apply to all same name alleles Allele color swatch displays the color for the selected allele Figure 6 8 Parameter Setting dialog box wt allele in group SNP4 selected 4 Two options are available when setting allele color If you want to apply the color to the same position number allele in all sae for example the first all
114. ed Values File 35KB Microsoft Excel Comma Separated Values File 35KB Microsoft Excel Comma Separated Values File 35KB Microsoft Excel Comma Separated Values File 26KB Microsoft Excel Comma Separated Values File J Ae Figure 6 16 MasterPlex GT window and Windows Explorer To open a project gtp drag the file onto the MasterPlex GT application window MasterPlex GT www miraibio com 6 17 CHAPTER 6 ALLELE CALL PARAMETERS 6 18 MasterPlex GT www miraibio com CHAPTER RESULTS TABLES The MasterPlexm GT software shows results in several tabular formats including the Typing table Allele Call table and Homology table This chapter explains how to view the tables and the data available in each 7 The Tvping Table In the Tvping table vou can view median fluorescence intensitv MFI relative intensitv RI or bead count data quickly assess assay performance designate negative controls compare results across experiments sort samples by expression level or genotype This section explains the different data views in the Typing table and its functional features Viewing the Typing Table The Typing table is the default view when you open a results file or project To display the Typing table for the active project click the button a particular project click Typing Table under the project of interest in the in the Project Manager Figure 7 1 MasterPlex GT www miraibio com 7 1 CHAPTER 7 RESULTS TABLES
115. ed and blue in the remaining tabs Figure 10 45 2 Double click the number in the Iv column MasterPlex GT www miraibio com 10 35 CHAPTER 10 GENOTYPING USING A LOOKUP TABLE Double click the lv number to display the inversion candidates 8 sho SS Bal Ee 8 or e mira Pee See eel a es es OO lt gt as dar Sta2 Eeldame feaa Mame Dash ftes eei dem fpf blank s200 No Matches off of 7 ei feom fess je JIET 7 al e l img O pot CO et e d a y l S M E E AANA 7 A l 1 a a jem em kmiem ea Di fiaooo_ gg fe Boro 5 27 S p E1 fioo fao of 5 8 Fa fiaooos_ sors No Matches ofj oj 3 1i Gi laoo tsa f Bom eoo F 1 Hi fiaoooz Jasso J oo Boro 5 27 a2 ios fasas J Boo Boost B2 Jisooos Issa f Mo Matches 9 2 d d a l U l 11 gp jim lI l c2 figooro Scot ot PI oj 8 II D2 aoon gst f oot Boo Fe 1s ease w N w on il heed o E2 figoor2 st fos Boro F2 fiona 39 oot soog F 20 n Inversion candidates for the B 001 B 003 genotype called for sample id0001 Figure 10 45 Typing table gradient background The Iv column shows the number of inversion candidates for a type Double click the Iv number to view the inversion candidates for a particular type 10 36 MasterPlex GT www miraibio com CHAPTER 10 GENOTYPING
116. ele in each group choose the option Apply to alleles in the same order in each group If you want to apply the color to all alleles with the same name in all groups choose the Apply to same name alleles option 5 Click Change Color The color palette appears Figure 6 9 6 10 MasterPlex GT www miraibio com CHAPTER 6 ALLELE CALL PARAMETERS e a BERET Lt ie BELL BERR Bi i i i i Basic ustom colors II l T an T Define Custom Colors gt gt Figure 6 9 Color palette 6 To select a predefined color click one of the basic colors 7 To define a custom color click Define Custom Colors The color palette shows the custom color options Figure 6 10 Basic colors ace E E hae E Ef Luminosity slider Eaa tm MA iH Custom colors Color swatch EEE EEE Ee ER EE EEE ee z nri Sat fi 86 Green 234 lefine Custe ColorlSolid Lum f150 Blue 1179 Cancel Add to Custom Colors Figure 6 10 Color palette Custom color options 8 To define a color use the click and drag operation to move the cross hairs in the custom color field Adjust the color brightness using the luminosity slider The Color swatch shows the color selection MasterPlex GT www miraibio com 6 11 CHAPTER 6 ALLELE CALL PARAMETERS 9 When you are finished defining the color click Add to Custom Colors to apply the color and click OK gt Ifyou selected a group
117. eles of the same locus group are represented by the same color If this option is not chosen each allele is represented by a different color The minimum number of beads that should be counted in the Luminex system for each bead type in each sample The ploidy may be specified for all groups loci or for individual groups The ploidy affects the allele frequency calculation Note Other and Haploid ploidy are the same The software calls the allele if all of the following conditions are met RI jee gt user specified RI threshold allele MFI jeje gt user specified intensity allele Z threshold The software calls the allele if both of the MEL ten gt user specified absolute intensity threshold NOTE If the bead count is less than the number of minimum 6 2 MasterPlex GT events specified in the Parameter Settings dialog box the Typing table displays the bead count data in red www miraibio com CHAPTER 6 ALLELE CALL PARAMETERS 6 2 Ploidy You can specify diploid haploid or other ploidy for each group locus The ploidy affects the allele frequency calculation see 4 e e Frequency on page 7 20 If you select diploid and there is only one allele called the allele will be shown twice in the Allele Call table i NOTE The Haploid option and Other option are the same 6 3 Relative Intensitv Allele Call For this method the MasterPlexm GT software computes the relative intensity RI of ea
118. erPlex GT www miraibio com CHAPTER 4 GETTING STARTED 4 Confirm the projects selected for the merge Remove the check mark from a project that you do not want to include in the merge To remove all check marks right click the wizard and select Uncheck All from the shortcut menu that appears To check mark all of the projects right click the wizard and select Check All in the shortcut menu 5 Click Accept to merge the projects and keep the Sample Merge wizard open Click OK to merge the projects and close the wizard Layer Merge Use the layer merge function to combine the results from different bead sets that probe the same sample For example after a layer merge the Typing table can display results from more than 100 different bead sets that probe the same sample NOTE To perform a layer merge a sample must have the same well location across all of the experiments and the bead names must be unique no two projects can include a bead with the same name lii m In the Project Manager right click the project of interest and select Project Merge Wizard from the shortcut menu that appears Figure 4 15 The Project Merge Wizard appears Figure 4 16 X Di HLA A B DR BLD Typing Table dl Multi Graph samples Typing view File Info dl Multi LI Sample2_ Typing all tulti Merge All Delete Project Merge Wizard LI Sample 5 Mik Typing Table dl Multi Graph LI SampleSmall Typing Table
119. eters Prefix Locus Name Ploidy Allele Narne RI MFI II Ir Other TJA 25 0 35 73G xl I3A 2 93G a Paie LLI gt Figure 11 8 Preview window 3 Click the Open Report toolbar button g The Open dialog box appears Figure 11 9 Look in 6 SampleData gt 4 fi ex Eg File name Files of type Report file frp sl Cancel Figure 11 9 Open dialog box 4 Specify a directory and enter the report name frp 5 Click Open gt The report is displayed in the Preview window MasterPlex GT www miraibio com 11 7 CHAPTER 11 REPORTS 11 8 MasterPlex GT www miraibio com APPENDIX APPLICATION OPTIONS The application options are user modifiable settings that are applied to all open results files csv or projects gtp This appendix explains the types of options available A 1 General Options 1 To view the general options select Options gt Set Application Options from the menu bar The Application Options dialog box appears Figure A 1 Bead Name Style e Locus Name Allele Name Original Bead Name Start Up Window After Data Loading Show Table view C Show Graph View Table View Gradation Background Use Allele Call Color for gradation background Group Color 1 aa Change Color Group Color 2 GW Change Color Heatmap Options Heatmap Bar Size f Pixel s f Allele Reset All Cancel Figure A 1 Application Options General tab In the General tab Fi
120. ety o ne nyo aj so C4 beadsold 951 Sample Empty l ii 7 82j ny 187 156 87P of nrj 188 j mj na MW2j 75 Isaj 126 o m n 1 88 135 o 183 1a eo Jasa an2 Samoe Emnty 18 61 182 e 72880600 120 38 7 86 72 tas 82 Bi Jaa 5063 JsamoleEmitv 108 58 148 138 167P 96 186 154 7 n dej s 88 t 82 IBID 188 E3 fara 462 Jsampletmotv 188 88 157 173 ta 88 183 148 D1 7 86048 123 88 t irm 167 189 99 2 185 1 63 169 ia 1235 86 139 128 9 e 13a 123 Figure 7 6 Typing table Bead count data 7 2 The Statistics Table In the Project Manager the Statistics table displays MFI background adjusted MFI and bead count data for user specified samples in the Typing table Figure 7 7 If you select more than one sample the Statistics table also displays the standard deviation SD and coefficient of variation CV 1 Do one of the following to select a sample s in the Typing table Click the sample name To select adjacent samples rows click and hold the mouse while you drag the mouse pointer over the sample names Click the mouse when the complete selection is highlighted Alternatively press and hold the Shift key while you click the first and last sample name in the selection To select nonadjacent samples press and hold the Ctrl key while you click the sample names MasterPlex GT www miraibio com 7 7 CHAPTER 7 RESULTS TABLES The Statistics table dis
121. f Group Name edit box Allele Name j Allele Call Parameters for SNP2 e Use Relative Intensity for Allele call Reportable Level 125 0 96 of total intensity Intensity Threshold 35 MF C Intensity based Allele Call Call anything bigger than fo MFI as an Allele Apply to all groups loci MFI Allele Ratio Figure 6 5 Parameter Setting dialog box Group name SNP2 selected MasterPlex GT www miraibio com 6 7 CHAPTER 6 ALLELE CALL PARAMETERS Parameter Setting Group set 7 Cancel Save setting as Import Setting Parameter setup for the individual bead Minimum Events ho count for each bead Use group color for Chart and Allele Call Table Lookup Table SNP1 SNPI Selected allele name ISNP3 SNP3 Diploid SNP4 SNP4 Diploid SNPS SNPS Diploid f Mi Group Allele Identifier Group Prefix of beads in this group 2 Edit Bead Names Group Name Ploidv f Dip Har Apply to all alleles in the Allele Name wt Change Color same order in each group Apply to all same name alleles Allele Name edit box Figure 6 6 Parameter Setting dialog box Allele name wt selected 6 6 Group and Allele Color If you choose the option Use group color for Chart and Allele Call Table in the Parameter Setting dialog box Figure 6 6 the MasterPlex GT software assigns one group color to all of the alleles in a group locus As a result the Multi Compare bar graph di
122. g the Y Axis Maximum 8 11 Threshold Editing lt 202sa0 449 54 eeea03 8 16 vili MasterPlex GT www miraibio com Changing the Intensity Thresholds 8 18 Sample by Sample Scatter Graph 8 19 Allele by Allele Scatter Graph 8 21 Copying a Graph ssibu ia bi iri ab bel bi pt we 8 23 CONTENTS Printinge a Gtaplies is ews w nra sie sr 8 23 Adding Graphs to a Report 8 23 CHAPTER 9 Cluster Analysis Displaying a Dendrogram 9 1 CHAPTER 10 Genotyping Using a Lookup Table Importing a Lookup Table 10 3 Creating a Lookup Table 10 6 Deine a Type sc0hsscmacdscageew ees 10 9 Setting Standards 92s i 2 4h 20s ben seetd 10 14 Specifying Cross talk 10 18 Setting General Parameters 10 20 Managing Lookup Tables 10 22 Editing a Lookup Table 10 22 Exporting a Lookup Table 10 24 Deleting a Lookup Table 10 28 HLA Typing Using a Lookup Table 10 29 Ambiguity Candidates 10 34 Inversion Candidates 0000 10 35 CHAPTER 11 Reports The Report Manager 2 22406 tesivszjjni 11 1 Adding Charts to a Report 11 2 Working With a Report in the Preview Window 11 3 Previewing a Report ess seci Kd heise se Ke oe 11 3 Searching a Report is susa miri paat jebdet dwa 11 4 gaing a Report xa saisb ai cesi raes 11 5 Print
123. gives the table background a striped appearance Uses a color gradient to indicate the relative expression level of the alleles called at each locus in a sample a lighter shade represents a lower expression level Alternating colors defaults are blue and red distinguish the alleles rows of the loci groups in the table Displays the dendrogram Displays the Multi Graph view for the active results Sorts the samples in the Typing table by homology to the expression levels of a user selected sample zi UF C 2 MasterPlex GT www miraibio com APPENDIX C TOOLBARS Table C 2 Typing table toolbar buttons and functions Typing Table Toolbar Button Function OF Resets the samples in the Typing table to the default order the order that sample data were acquired in the Luminex system Show the Typing table with a separate tab for each locus Displays a Type column for each locus in the Typing table C 3 Multi Graph View Toolbar Sjo a ME nee xl SE err wao vaol Figure C 3 Multi Graph view toolbar Table C 3 Multi Graph view toolbar buttons and functions Multi Graph View Toolbar Button Function Opens the Parameter Setting dialog box for the active results csv or gtp oq Displays 3 dimensional bars in the Multi Compare graph Th Displays allele name tags in the Multi Compare and Depth bar graphs Displays a legend of allele names for the Multi ii Compare and Depth bar graphs Dis
124. gure 8 5 Multi Compare graphs MFI data Adjusted MFI of 47 1 B1 A IB Anderson 308 NA ae on bakes Na II PA AE DAW A A IB Ic Ic2 ID 1841142 IB lic IID Adjusted MFI of 48 1 F2 or ka OM SM MEM ME ILA AW AT d A B 1 C2 ID 141142 IB IC ID Adjusted MFI of 48 2 G2 W 49 A WA SEES L A IB Ic IC2 ID 181142 IB IIc IID MasterPlex GT www miraibio com 8 7 CHAPTER 8 GRAPHS JEF Multi Compare Th Depth EE Threshold Editing EE Sample by Sample E Allele by Allele Relative Intensity ot 47 1 81 100 Anderson 1A 161240 161260 go 161294 ta mile p A e Anderson mija 18 49 Bore FF FF a 16217C a 40 162237 fli ban E U ER Bo oe 182240 L 20 Anderson iE 1 Far ff K aa eaa 4 IE D IA1 2 IB IC IC Relative Oooo a C2 100 1 1 i i gt fi i Dy Oe Anderson A A IB nali rs Ree A Oni onl 18126 ie i ae 1 H 16129A s a z i Anderson 5 ote 463100 16320T ote eee ee E MT 162237 THEa B 08 08 UB e g 182240 L 20 TM Anderson s h IIL ten 0 FI EEA CLM 162927 16205T 3 ae ID Wd WA IB IZ IIC 16294T 11 Relative Intensity of 45 4 AS IET 100 4 rot OE 161240 a ee 161262 g0 16129A pop hii e gaa aya ra ta E A cyt Anderson if BOs IB i B E omy a error cae i l 16217C p 40 i 182237 JW EEEa d EP B i i enn Sai MMA li 182240 L 20 Anderson th ja ju iet a l A F L a ja a n 162027 162957 13 C1 D hd
125. gure A 1 the user modifiable settings include Bead Name Style Specifies how the bead set names are displayed in the Typing table Choose the locus and allele names or the names entered in the Luminex system Start Up Window After Data Loading Determines whether the Project Window displays the Typing table or the Multi Graph view upon opening a results file csv or a project gtp MasterPlex GT www miraibio com A 1 APPENDIX A APPLICATION OPTIONS Table View Gradation Background Enables you to choose differ ent gradient background colors for the Typing table Heat map Options Enable you to change the width of the bars in the map Bead Name Style You can specify how to display the bead names in the Typing table Figure A 1 Choose one of the options Locus Name Allele Name displays the locus group name and the allele name for each bead type Figure A 2 Original Bead Name displays the bead name entered in the Luminex system Figure A 3 Locus name first row and allele names next row po totus Asner dea f ses spa tt fas wt wt mt wt m Well Name JSample Name Total Events Notes _ a a c 50 0 800 www 7 at ooma a i d Figure A 2 Typing table Locus Name Bead Name option selected SS SS Se a ener eneit enez oo fenes wa fena m enea wa enean enes we JG IET TE A SSE JE EIST PSR RC SRS S JPEEEZIJEREJI PSE ce Poe of 0 8 3 z Figure
126. he haplotype frequency information right click the table and click Copy Table as Text from the shortcut menu that appears The table information is copied to the system clipboard Allele Call Sample2 Sample2 gtp pale 1 m 3ij XI Allele Call Allele Frequency Genotype Frequency Haplotype Frequency Anderson Anderson 16311C 16311C 16320T_ 16362T Anderson 16294T Anderson 16362T Anderson Anderson 16126C Anderson Anderson 16217C Anderson 73G 934 152C 195C 263G 734 93G Anderson 195C 263G 195C 263G aon U We U N Gf Figure 7 21 Allele Call table Haplotype Frequency tab 7 22 MasterPlex GT www miraibio com CHAPTER 7 RESULTS TABLES 7 5 Homology Table and Chart The MasterPlex GT software computes a homology score for each pair of samples in the Typing table It applies a least squares analysis to the expression level of the alleles MFI data for each sample pair The Homology table displays the homology scores for the sample pairs Figure 7 22 and the Homology chart plots the data in 3 dimensional space Figure 7 23 1 To view the Homology table click the a button A separate window opens and displays the Homology table Figure 7 22 In the Homology table the scores are colored white no homology 0 blue perfect homology 1 and shades of red a darker shade represents a larger homology score E Homology Plot Sample 0 46368 0 40198 0 33525 0 0 30469 0 49
127. he radio button next to the sample you want to use as the reference gt Alleles that are called different from the reference are painted using the group or allele color depending on what is selected in the Parameter Setting dialog box See Group and Allele Color on page 6 8 3 To toggle the view and paint the alleles that are called the same as the reference click the 2 button Figure 7 15 7 16 MasterPlex GT www miraibio com CHAPTER 7 RESULTS TABLES wt wt WE WE WE WE WE Wwe WE wE WE tajt mit rat WE wt mit rat Figure 7 15 Allele Call table Alleles called the same as sample 6 user selected reference are painted 4 To indicate samples that have fewer alleles called at a locus than the reference sample click the button The Allele Call table displays for samples that have fewer alleles called at a locus than the reference sample Figure 7 16 FI Allele Call Sample1 Sample1 gtp Allele Call Allele Frequency Genotype Frequency Haplotype Frequency Ir IA IB IC1 IC2 ID IIB IIC 16223T 16292T 16295T_ 16311C 163627 16223T 16292T 16295T 16311C 16362T 16223T 16292T 16295T 16311C 16223T 16311C 16362T 163194 16223T 16311C 16320T 16223T 16311C 16362C 16311C 16320T 16223T T 16311C 16362T 162231 Anderson 16362T Reference D7 h16401 1 sample F7 h16401 3 r G9 hi6401 20 r G8 hi6401 12 lt lt lt lt
128. how Table View C Show Graph View Table view Gradation Background Use Allele Call Color for gradation background Group Color 1 Change Color Group Color 2 Gw Change Color Heatmap Options Heatmap Bar Size 3 Pixel s Allele Reset All Cancel Figure 3 3 Application Options dialog box General tab 2 To organize bead names in the Typing table by group and allele name Figure 3 1 choose the Locus Name Allele Name option 3 To organize bead names in the Typing table in the order that the data were collected Table 3 2 choose the Original Bead Name option 4 Click OK Editing the Bead Name The prefix set in the Luminex software is the default locus group name in the MasterPlex GT software Figure 3 1 You can edit the prefix group or allele name for a bead in the Parameter Settings dialog box or the Typing table To rename a bead in the Parameter Settings dialog box 1 Click the Parameter Setting button ah The Parameter Setting dialog box appears Figure 3 4 3 4 MasterPlex GT www miraibio com CHAPTER 3 BEFORE YOU BEGIN F Parameter Setting Group set Cancel Save setting as Import Setting V Parameter setup for the individual bead Minimum Events 20 count for each bead IV Use group color for Chart and Allele Call Table Lookup Table Prefix Group Name Type Lookup Table Allele Name Reportable Level Intensity Threshold Call Intensity
129. iles HitachiSoft MasterPlex GT 2 0 Settings mas L frf ja 10 1 2002 9 59 AM eas P frf 3 WW FRFFile 10 1 2002 9 59 AM File and Folder Tasks Other Places y ad L if KB FRFFile 10 1 2002 9 59 AM E FRF File 10 1 2002 10 00 AM FRF File 10 1 2002 10 00 AM FRF File 10 1 2002 10 00 AM GTT File 10 31 2003 12 56 PM FRF File 10 1 2002 10 00 AM FRF File 10 1 2002 10 01 AM FRF File 10 1 2002 10 01 AM FRF File 10 1 2002 10 14 AM E MasterPlexGT gto 4KB GTOFile 10 31 2003 4 37 PM Figure 10 1 Steps to type samples using a HLA lookup table Steps continued in Figure 10 2 MasterPlex GT 3 www miraibio com 10 1 CHAPTER 10 GENOTYPING USING A LOOKUP TABLE 3 Set thresholds or select a group set Group set I E Choose Intensity based allele call sors or ote Parameter setup for the individual bead Minimum Events 20 count for each bead Call anything bigger than 50 MFI an allele Eeee mersanaeca rue eed Pretix Group Name Type Lookup Tabie Report jintensav Call inten A Click Apply to all groups Group Aliele Identifier Group Prefix of beads in this group 7 Group Neme JA Change Coine Prokty C Diploid C Hoploid Other _ Apply this Pioidy to al groups ci _ Allele Noene 1 change ooe Ie Allele Call Parameters for A Use Relative Intensity for Allele call Reportable Levet of total intensty Call anything bigger then 50 MFI as an Allele Apply to ail groups loci 4 View
130. ing a Report i465 ia emee o s 11 5 Opening a Report t2t4anoiiancsemnteaats 11 6 APPENDIX A Application Options Gentral OPOONS ssir fib oxaeernedaneeas A 1 Bead Name Styles s snbsessato isitta sea A2 Start Up Window After Data Loading A2 Table View Gradation Background A 3 Heat Map Options ssssaint teen t ena A 6 Background Options 00 A 6 Clustering Tool Options A 8 LA in iet Gating e ae Eas A 9 Resetting the Application Options A 10 MasterPlex GT www miraibio com ix APPENDIX B Project Options amp Parameters CONTENTS Allele Call Parameters 0 000 cee eues B 2 Allele Call General Options B 2 Allele Call Parameters B 2 Table VEW iso is esti 050 edd bii esi ox B 4 Value to Show eee B 4 Cell Background Coloring Mode B 5 GHD VieWs th b A O ad B 6 Allele Call General Options 21ic0ccaiues B 7 Allele Bar Graph Options B 7 Allele Call View 0 ccc cece eee eee B 12 Allele Call Table Background Coloring B 14 Cluster Anglys Sir srl snien tt bj B 17 Resetting the Default Parameters B 17 APPENDIX C Toolbars Main Toolbar ss rivesssssdsszj s esa sjz C 1 Typing Table T oblbat lt 2020 042426008 men C2 Multi Graph View Toolbar C 3 x MasterPlex GT www miraibio com CHAPTER WELCOME MiraiBio MasterPlex M GT Welcome to the MiraiBio MasterPlex GT User Manual MasterPlex GT softwa
131. ions 8 10 8 15 printing 8 23 Sample by sample scatter 7 27 Sample by Sample scatter graph 8 19 8 21 MasterPlexGT www miraibio com I 1 group color 6 8 6 12 group name editing 3 4 6 6 group set creating 6 12 importing 6 13 selecting 6 13 H haplotype frequency 7 22 hardware requirements 2 1 heat map options A 6 HLA typing 10 29 10 33 overview 10 1 10 2 Homology table and chart 7 23 I import lookup table 10 3 10 5 installing MasterPlex GT 2 1 2 4 license 2 3 intensity based allele call 6 2 intensity based allele call 6 5 inversion candidates 10 35 L license 2 3 information 2 4 lookup table copying 10 28 creating 10 6 10 21 define type 10 9 10 14 deleting 10 28 editing 10 22 10 24 exporting 10 24 10 27 importing 10 3 10 5 set general parameters 10 20 10 21 set standards 10 14 10 17 specifying cross talk 10 18 10 20 Lookup Table Editor 10 6 M MasterPlex GT analysis overview 3 8 3 19 minimum events 6 2 Multi Compare graph 8 6 options 8 10 8 15 Multi compare graph 7 25 Multi Graph view 4 5 8 1 Multi Compare graph 8 6 sorting by expression level 8 3 negative controls global 5 1 5 2 identifv A 6 local 5 1 5 2 removing 5 3 setting automaticallv 5 3 setting manuallv 5 2 O overview 3 8 3 19 HLA tvping 10 1 10 2 overview of analvsis 3 8 3 19 P ploidy 6 2 6 3 plug ins A 9 print graph
132. is displayed in a separate Project Window Table 4 1 on page 4 4 shows the options available to you for displaying the Project Manager and Project Windows MasterPlex GT www miraibio com 4 1 CHAPTER 4 GETTING STARTED L Sample Typing Table dl Multi Graph DI SampleBase Typing Table dl Multi Graph O SampleSmall Typing Table dl Multi Graph Project tree MFI Adjusted MFI Count cv i 0 68 10 2 os 91 DE 15 7 35 BAX 241 14 1 53 73 16 2 11 0 38 12 15 417 Em gt Statistics table Figure 4 1 Project Manager The Project Manager includes two components a tree of open results and a Statistics table for samples selected in the Typing table 4 2 MasterPlex GT 3 www miraibio com CHAPTER 4 GETTING STARTED II Typing Sample2 Sample2 gtp O x jeb bial 5 jie SSS SSS eS Sa a SS a SS e ES SS Sara ao Ia heze ret26 161208 andersor 16217 162237 16224C_ andersor 162927 1 WellName Samole Name TotalEverts Notes TT TT S i l SESA GI TE C71 7 Sample Emnty 141 61 125 305 3 48 1d 630524 Sample Empty 10 130 65 112 287 i 1 Ai _ beads onlvnew __ 2253 Samole Emnaty z z z 7787 Sample Empty gt z z beadsold st Samole Emnty G 3 a a i x Be o kwa dt Samole Ematy 402755 ais 65 135 20d 2 smo aa lang Samoe Emoty A 0258 82187075 165 207 0 clan tg Samole Ematy 1 7 s a nN w 5 47 2d 8115
133. l Figure A 7 Application Options General tab Heat Map Options The default bar width is 6 pixels To change the bar width make a selection from the Pixels allele drop down list 1 pixel minimum width 10 pixel maximum width A 2 Background Options The MasterPlex GT software can automatically identify the negative controls in a results file csv by searching for key words in the sample name In the Background tab of the Application dialog box you can set the key words that identify a negative control 1 Select Option Set Application Options from the menu bar The Application Options dialog box opens Figure A 8 A 6 MasterPlex GT www miraibio com APPENDIX A APPLICATION OPTIONS Application Options Automatic Background Sample Recognition Iv Perform Automatic Background Sample Detection on data load Background Keywords bead only Add New Keyword beads only bka background blank NOTE These parameters are effective only to new projects Reset All Cancel Figure A 8 Application Options Background tab Click the Background tab 3 Choose the option Perform Automatic Background Sample Detection on data load 4 To define a keyword click Add New Keyword enter the key word s in the dialog box that appears and click OK Figure A 9 The keyword is added to the Background Keywords list Figure A 8 NOTE A keyword added during a session is applied only to a subseque
134. le Selection window click B to open the Parameter Setting dialog box and click Lookup Table Lookup Table Selection Entry Name Last Modified EEA EM A PROJECT HL44 HLB HLADR Blood Type verl i0 VWerl 10 Veri1 10 Yeri o0 Create Mew Lookup Entry HLA Lookup Table 10 31 2003 12 56 50 PM IHLA A IHLAB HLA DR Blood Type Delete Selected Entry Select Latest Tables Unselect All Cancel Figure 10 36 Lookup Table Selection window 2 Click the table row that you want to delete and click Delete Selected Entry Click Yes in the confirmation message that appears 10 28 MasterPlex GT www miraibio com CHAPTER 10 GENOTYPING USING A LOOKUP TABLE 10 4 HLA Typing Using a Lookup Table After you open a Luminex results file csv and import or create a lookup table the steps to type a sample include Set parameter settings Select a lookup table View the Typing table 1 To open a Luminex results file csv click the Open CSV File but ton LI Alternatively select File gt Open CSV File from the menu bar The Open dialog box appears Figure 10 37 2 Select the csv of interest and click Open The Typing table displays the results data Hi samplel es l H Jsample csv 85 SampleBase csv 8 Samplesmall csv File name IHLA 8 B D F_BLD cev Files of type Lumines Data file cev mdb Cancel Figure 10 37 Open dialog box MasterPlex GT www miraibio com 10 29 CHAPTER 10 GENOTYPING U
135. leEmetv C4 beadsold ee Empty p21 JsampleEmetv Ba ama lesa Sale Eimnty Boo aa S51 Seale Eimnty Eoo o faa 5063 Sale Eimnty 2 48 4 7017 Sample Empty 48 3 5242 Sample Emoty EJT T Ttaitaitaoa man L nr ro N N pol Oo o a wi m m m an oD a YH OH BH BY Nl YO KH amp WO LI ny ee Oo UO Nal Nal HO BH WwW c NDAN Pr On amp amp WMH HH Ws v ww awn nnn Figure 7 5 Typing table Background adjusted MFI data Bead Count View In this view the Tvping table displavs the number of events beads that the Luminex svstem counted for sample acquisition e To display the bead count view click the Y button The Typing table displays the bead count data for each allele Figure 7 6 NOTE Data displayed in red indicates the bead count was less than the user specified event number set in the Parameter Setting dialog box 10 To view the MFI and percent relative intensity data position the mouse pointer over the table cell of interest 7 6 MasterPlex GT www miraibio com CHAPTER 7 RESULTS TABLES A pop up tool tip displays the MFI and percent relative intensity for the allele lof fE 2a Ee aa enn ee Es En es ee A EE ae JUL es ae Lae ose ita selene jw ane Jaage buses td GIAN RAW E a 8 ooj eej eej 17 105 tie t22 66 188 118 Fa fae Je305 Sample Em
136. lele Call Table Lookup Table Lookup Table Allele Name 96Report Intensity Call Inten A Allele name Diploid 16124C 25 0 US 1 62 1 7C 16126C 25 0 selected 161294 25 0 Anderson 25 0 16217C 25 0 16223T 25 0 16224C 25 0 Anderson 25 0 IC1 Ic1 v ADANITALCANET Ac mow lt i a Group Allele Identifier x tt P To edit a Group Prefix of beads in this group 4 Edit Bead Names group name Group Name OS Change Color select a grou g p Ploidy U jw e name above and edit the Allele Name 16217C l name here B shele Call Parameters for IB 16217C To edit an al lele e Use Relative Intensity for Allele call name select an Reportable Level 25 0 of total intensity allele name intensity Threshold 35 MFI above and edit the name here C Intensity based Allele Call Call anything bigger than MFI as an Allele Apply to all beads Figure 3 8 Parameter Setting dialog box 2 Edit the name in the Group Name or Allele Name box 3 Click Apply The Parameter Settings dialog box displays the new name 3 2 Overview of MasterPlex GT Analysis This section provides an overview of MasterPlex GT genotype analysis An analysis workflow includes the following steps 1 Open the results files csv of interest and view the Project Man ager and Project Window Figure 3 10 A separate Project Window opens for each results file and displays the intensity data in the Typ ing table Figure 3 10
137. me that the MasterPlex GT software automatically assigns to each bead The Typing table sorts the bead names by group locus name then by allele name Figure 3 1 This enables you to plan a bead naming strategy that optimizes the Typing table view for your needs Table 3 1 summarizes the MasterPlex GT bead naming conventions MasterPlex GT www miraibio com 3 1 CHAPTER 3 BEFORE YOU BEGIN Table 3 1 MasterPlex GT bead naming conventions Name Component Specified Identifies the Prefix By the user in the Luminexe Gene region or software The prefix can be marker that the edited in MasterPlex GT probe interrogates Group locus Automatically by the Gene region or name MasterPlex GT software The marker that the prefix is the default group probe interrogates name The group name can be edited in MasterPlex GT Allele name By the user in the Luminex Variation that the software The allele name can probe interrogates be edited in the MasterPlex GT software Table 3 2 shows example names for the beads that interrogate human mitochondrial DNA at Hypervariable Region LA Figure 3 1 shows how the Typing table organizes the bead names by locus group and allele name If the bead names do not follow the MasterPlex GT naming convention the Typing table displays them in the order that the data were collected Figure 3 2 Table 3 2 Example group and allele names for beads that interrogate human mitochondrial DNA at
138. mp PARAMETERS B 5 Cluster Analysis Default Cluster Analysis Make a selection from this drop down list to Method set the default cluster analysis tool Cluster Analysis By Choose this option to cluster samples Expression according to the MFI data Cluster Analysis By Choose this option to cluster samples Genotype Haplotype according to the genotype of all the alleles F New Project Default Parameters Default Cluster Analysis Method Ward s Method x Parameter for Flexible Method 0 25 gt Default Cluster Analysis Options Cluster Analysis By Expression e Cluster Analysis By Genotype Haplotype OK Reset All Cancel Figure B 15 New Project Default Parameters Cluster Analysis tab B 6 Resetting the Default Parameters To reset the options and parameters to the default factory settings click Reset All Figure B 15 At the prompt click OK MasterPlex GT www miraibio com B 17 APPENDIX B PROJECT OPTIONS amp PARAMETERS B 18 MasterPlex GT www miraibio com Me oman C 1 Main Toolbar Deal SIHEJ Hal S Ale BIBIM Figure C 1 Main toolbar Table C 1 Main toolbar buttons and functions Main Menu Bar Toolbar Command Button Function File gt Open Displays the Open dialog box so that CSV File a Luminex results file csv may be opened File Open Displays the Open dialog box so that Project File a project gtp may be opened File gt Save Displays the Save As dialog box so Project that a
139. mplete window 5 Click Finish 2 3 Installing a License 1 Double click the MasterPlex GT icon Pi on the workstation desktop The License Information dialog box appears Figure 2 4 MasterPlex GT www miraibio com 2 3 CHAPTER 2 INSTALLING MASTERPLEX GT License Information E E xl Product Hame MasterPlex GT version 27 0 0 Build 185 User Name Institute Division Number of Licenses Date lssued Licensed Version Licensed Machine ID Expiration Date Machine ID of this PC 2E 71 628 3 30BE Obtain New Licenses Demo Licenses Close Figure 2 4 License Information dialog box 2 To view instructions on how to obtain a license lic click Obtain New Licenses 3 After you have obtained a license click Install New License The Open dialog box appears 4 Use the Open dialog box to locate the license lic and double click the file The license is installed 5 To view the license information select Help gt View License Info from the menu bar gt The license information is displayed Figure 2 5 License Information xl Product Marre MasterPlex GT version 2 0 0 Build 185 User Mame Katherine Shigekawa Institute Cogent Comm Division Number of Licenses 1 Date Issue 20034 0 29 Licensed Version version 2 0 0 Build 0 Licensed Machine ID 461 0 5966 7685E Expiration Date 20034 2 268 Machine ID of this FE 461 0 5986 7685E Obtain Mew Licenses Dem
140. n the Multi Compare and Depth graphs Displays a drop down list of size options for the vertical dimension of the bars in the Multi Compare and Depth graphs www miraibio com MasterPlex GT A Allele by Allele scatter graph 8 21 8 22 allele call intensity based 6 2 intensity based 6 5 relative intensity 6 2 6 3 6 5 allele call parameters group and allele color 6 8 6 12 settings and options 6 1 6 2 B 2 B 3 Allele Call table 7 11 setting defaults B 12 B 13 sort bv expression level 7 18 allele color 6 8 6 12 allele frequency 7 20 allele name editing 3 4 6 6 ambiguity candidates 10 34 bead name conventions 3 1 3 4 editing group or allele name 3 4 6 6 options 3 4 A 2 C cluster analysis 9 1 9 4 selecting default method B 17 tool options A 8 controls global negative 5 1 5 2 local negative 5 1 5 2 removing 5 3 setting automaticallv 5 3 setting manuallv 5 2 copy graphs 8 23 cross talk define in lookup table 10 18 10 20 D dendrogram 9 1 9 4 Depth bar graph 7 25 Depth graph 8 8 options 8 10 8 15 E editing allele name 3 4 6 6 group name 3 4 6 6 G general parameters define in lookup table 10 20 10 21 genotype frequency 7 21 graphs Allele by Allele scatter graph 8 21 8 22 copying 8 23 Depth 8 8 Depth bar 7 25 Depth options 8 10 8 15 display options B 6 Multi Compare 8 6 Multi compare 7 25 Multi Compare opt
141. neral Figure 10 26 Lookup Table Editor Cross talk view In Figure 10 26 the software computes MFI o1 corrected for cross hybridization as follows Corrected MFI o1 Original MFI 9 0 03 MFT 92 0 02 MFT 05 7 To delete an allele from the cross talk table click the row that you want to delete and click the Delete button T Alternatively right click the Lookup Table Editor and select Delete from the shortcut menu that appears Click Yes in the delete confirmation message that appears 8 To reset all cross talk entries to zero default click Reset To enter cross hybridization values for another allele follow step 3 to step 6 10 To enter cross hybridization values for the alleles at another locus follow step 1 to step 9 Setting General Parameters In the General view of the Lookup Table Editor Figure 10 17 you can specify The allele ploidy The text that the Typing table displays when a sample has no matching genotype Whether to display standard data in the Typing table 1 Inthe Lookup Table Editor click a tab to select a locus 2 Click the General button CJ 10 20 MasterPlex GT www miraibio com CHAPTER 10 GENOTYPING USING A LOOKUP TABLE The General view for the selected locus or blood type appears Figure 10 23 al Lookup Table Editor Export Apply Reset All O4 O8 Oor O eo Locus Setting Name HLA A Version h 4 Group Name Task Parameter
142. ng Table P id TEL Multi Compare Ta Depth EE Threshold Editing EE Sample by Sample Allele by Allele dl Multi Graph Ki C SampleBase l BOE U NI Adjusted MFI of 1 B1 Typing Table CCl Tn Se eee aaa eae il MultiGraph 2 n3 NI gi D Sampei SEEM ee Pee eer eee eee eee eee ees eee ieee eae ne hers Typing Table Fl 5 Poa ah me il Multi Graph i ti TB Ma ee eee mae are CS ener ee eee eee eee ene O Sampe BIT EEMTR jA Typing Table e TEE EN KA ola af ar ara ae Be ee t il MultiGraph O sid TA SNP1 SNP2 SNP3 SNP4 SNPS SNPS SNP7 SNP amp SNP9 SNP10 Adjusted MFI of 4 E1 x 1 500 MFI Adjusted MFI Count 3 54000 f 75 4 a 59 7 500 424 317 6 75 0 z 59 36 6 61 6 707 559 7 58 29 5 79 2 51 2 wy Adjusted MFI of 8 42 wes 492 402 9 91 8 SNP 73 26 3 35 9 amt 282 398 8 137 9 SNP2 297 449 8 151 5 372 535 4 143 9 236 418 4 141 5 1085 871 2 53 16 9 1032 818 9 124 36 4 277 211 5 80 3 31 9 79 4 URA er Max 1731 AUTO MFI Background EE Project Manager Project Window displaying includes file tree top the Heat map and Multi and Statistics table Compare graph bottom Figure 3 11 Project Manager and Project Window Multi Compare graph in the Project Window Sample Name T co 07 on fF oo Pa oO Fa oo EERE wk MFI 586 90 3 Figure 3 12 Heat map MasterPlex GT www miraibio com 3 13
143. ntly opened results files csv or projects gtp Add a Background Keyword Input a newe keyword for background canoe Figure A 9 Add a Background Keyword dialog box MasterPlex GT www miraibio com A 7 APPENDIX A APPLICATION OPTIONS 5 To delete a keyword select the keyword you want to delete in the Background Keywords list and click Delete Selected Keyword At the prompt click Yes A 3 Clustering Tool Options You can have the Clustering Tool window Figure A 10 open in the Multi Graph view when you work with the Multi Compare Depth or Sample by Sample scatter graph You can adjust the transparency of the Clustering Tool window NOTE This option is only available for the Windows 2000 or XP operating system SampleSmall Clustering Tool N N Min F N Max Median Centroid pa B G Link Ward s Flexible Genotype Expression Figure A 10 Clustering Tool window 1 Select Option Set Application Options from the menu bar The Application Options dialog box opens Figure A 11 Application Options Clustering Tool Window Transparency 15 NOTE Transparency is effective only on indows2000 XP platform Reset All Cancel Figure A 11 Application Options Clustering Tool tab A 8 MasterPlex GT www miraibio com APPENDIX A APPLICATION OPTIONS 2 Click the Clustering Tool tab 3 To increase or decrease the Clustering Tool window transparency click
144. o Licenses Close Figure 2 5 License information 2 4 MasterPlex GT www miraibio com CHAPTER BEFORE YOU BEGIN This chapter explains the MasterPlex GT bead naming conventions It also provides an overview of genotype analysis using the MasterPlex GT software 3 1 Bead Name Conventions The MasterPlex GT software organizes results data csv by bead and sample name in the Typing table Figure 3 1 See The Typing Table on page 7 1 for more information Further the software can combine or merge results that are derived from identically named bead sets or merge the results from different bead sets that probe the same sample The merged data are displayed in one Typing table enabling you to analyze and compare samples across experiments or view the results from more than 100 different bead sets per sample See Merging Results on page 4 11 for more information The MasterPlex bead naming convention includes a group and allele name Table 3 1 To follow this naming convention in the Luminexe data collection software specify e a prefix that identifies the gene region that the probe interrogates For example the prefix can identify a locus intron or exon or other marker see Table 3 1 and Table 3 2 e an allele name that identifies the variation at the gene region that the probe interrogates For example the name can identify an allele or a SNP base see Table 3 1 and Table 3 2 The prefix is the default group na
145. of interest onto the MasterPlex GT application window 4 10 MasterPlex GT www miraibio com 4 3 CHAPTER 4 GETTING STARTED Merging Results You can combine or merge results and view the merged data in one Typing table There are two ways to merge results Sample merge Merges the results from different samples that are probed by identically named bead sets See page 3 1 for more infor mation about MasterPlexm GT bead name conventions A sample merge enables you to apply the same controls across experiments compare control data and compare results across experiments Layer merge Merges the results from different bead sets assays that probe the same sample If an assay format distributes the same sample across several different wells and probes each well with a different bead set you can use the layer merge function to view the results from the different bead sets in one Typing table Sample Merge Use the sample merge function to combine the results from different samples that use identically named bead sets There are three ways to perform a sample merge Drag and drop method Batch method Merge wizard NOTE Only results from identically named bead sets can be merged using the drag and drop method Sample Merge Using the Drag and Drop Method 1 In the Project Manager click and hold the file of interest while you drag it to the file that you want to merge it with Figure 4 10 MasterPlex GT
146. okup Table Editor Reset All Removes all changes from every locus Reset Removes the changes from the currently selected locus Cancel Closes the Lookup Table Editor without applying any changes Defining a Type The Type view is the default in the Lookup Table Editor Figure 10 10 In this view you can specify or edit the allele expression pattern that defines a type 1 Inthe Lookup table Editor select a locus tab Figure 10 11 2 Enter a Locus Setting Name For example in Figure 10 10 the A tab is selected and HLA A was entered for the locus setting name The Lookup table Selection window will display this name 3 Enter a version number for the selected locus or blood type Figure 10 11 MasterPlex GT www miraibio com 10 9 CHAPTER 10 GENOTYPING USING A LOOKUP TABLE 1 Click a tab to 2 Enter a name for the 3 Enter a version number for the select the locus selected locus or blood type selected locus FI cwokup Table Editor Export Apply Reset All 4 08 Oor O er a Zo Locus Setting NameCHLA A Version 1 1 D Group Name Reset Type Standard Beads Cross Talk The locus setting name and version number entered in the Lookup Table Editor top will appear in the Lookup Table Selection window bottom BI Lookup Table Selection T mg Edit Selected Entry Fi Create Mew Lookup Entry Ver 10 ver1 10 Veri l0 verl 00 Delete Selected Entry Select Latest
147. ols can be applied to a results file The MasterPlex GT software first determines if global negative controls have been specified and computes Global NC Global NC Global NC Global NC n Next the software examines each results file for local negative controls local NC The background value for a results file is the average of the local NCs and global NC Table 5 1 on page 5 2 shows example results files and negative controls and how the MasterPlex GT software computes the background value for each results file In this example Global NC Global NC Global NC Global NC 3 The MasterPlex GT software subtracts the computed background value from the sample bead set MFI to obtain the background adjusted sample MFI data MasterPlex GT www miraibio com 5 1 CHAPTER 5 NEGATIVE CONTROLS Table 5 1 Computed background values Local Global Results Negative Negative Computed Background Value Controls Controls Platel Local Local NC Local NC Global NC NC 3 Local NC Plate2 Local Local NC Global NC 2 NC Plate3 None Global NC Plate4 Global NC Global NC Plated Global NC Global NC Global NC a Global NC Global NC Global NC Global NC 3 5 2 Setting Negative Controls Manually If you are working with one set of results you can set a oca negative control If you are working with merged results you can set a global negative control that is applied to all of the merged
148. ottom MasterPlex GT www miraibio com B 11 APPENDIX B PROJECT OPTIONS amp PARAMETERS B 4 Allele Call View In the Allele Call View tab you can specify defaults for the Allele Call table FI New Project Default Parameters Genotype Lising Direction e Vertical C Horizontal Allele Call Table Background Coloring C Paint alleles with color if genotype is same as the reference e Paint alleles with color if genotype is different from the reference Allele Call Table Options IV Enable Reference Sample Selection M Show when number of alleles is fewer than the reference OK Reset All Cancel Figure B 11 New Project Default Parameters Allele Call View tab Genotype Listing Direction Vertical Choose this option to display genotype calls in a vertical list in the Allele Call table Figure B 12 Horizontal Choose this option to display genotype calls in a horizontal list in the Allele Call table Figure B 12 B 12 MasterPlex GT www miraibio com APPENDIXB PROJECT OPTIONS amp PARAMETERS Allele Call Sample2 Sample2 gtp Allele Call Allele Frequency Genotype Frequency Haplotype Frequency F1 48 1 r F2 48 1 16126C Anderson f F3 48 1d 16126C Anderson C4 beadsold f B2 47 1 r B3 47 1d f B1 47 1 Anderson Anderson Anderson Anderson Anderson I6217C Anderson Anderson 16311C 16311C 16320T Anderson 16311C 16311C 16320T Anderson 16311C 16311C 1
149. ound Figure 7 3 To view the Typing table with the striped background click the button gt The alleles columns associated with a locus appear blue or white in the Typing table Figure 7 2 To view the Typing table with the gradient background click the button The Typing table highlights the allele relative intensity using a color gradient Figure 7 3 A darker color shade indicates a higher relative intensity Red and blue distinguish between the loci Alleles at locus IA Alleles at locus IB sample Emoty 1 0 l sample Emoty 1 1 l sample Emoty 1 1 oj Sample Empty ki TJ T 7 7 Sample Empty ii a i T B z ca beadsold Sample Emoty gt ka l z B2 aa fes Sample Emoty 5 5 d MMA 65 Ba sd s894 Sample Emoty 5 6 J ike 74 Boo o sa 5519 JSampieEmetv l 4 5 B 7 75 caren 8 JSampleEmetv 9 1 d 75 ar c2 47 2 s409 Saamiple Emetv 2 A 2 2 74 39 cea Jaza ft JsampleEmetv 1 2 2 0 69 45 7 195 ot o ea 5122 JsampieEmotv 9 3 1 1j 60 44 im e2 fae S135 JsampieEmotv 1 4 2 58 45 SAMA Ltd os fapezg 5012 JsampieEmetv 9 1 1 2j 48 38 Em eio o 4740000000 50830 JSampieEmetv 1 0 2 1 1 1 1 1 a2 ea J7017 000 JSampleEmetv 2 1 1 21 1 1 2 2 e2 aa 3109 JsampleEmetv 9 0 2 1i l 0 l i a3 00 ea o enm Semple Emoty 9 1 1 ej 3 1 2 2 E3 la7ead BAD JSampieEmetv 2 1 2 ui 2 0 1 2 48 4d Sample Emoty 1 1 2 2 2 0 1 2 oO are JSampleEme
150. plays the average MFI for the selected samples standard deviation SD and coefficient of variation CV of the MFI background adjusted MFI and bead count data for the selected samples Figure 7 7 NOTE The Statistics table is automatically updated when you select new samples in the Typing table LI samplet Typing Table il MultiGraph File tree D sample Typing Table dl Multi Graph MFI Adjusted MFI Count Bead Ave SD cyz 1 5 130 9 1 0 200 0 Project 0 6 346 4 Manager Le 1378 ______ Statistics table 3 8 7 8 5 5 5 0 0 6 0 8 1 8 0 6 4 8 1 0 400 0 06 20 4 46 78 16 8 40 46 11 6 258 200 7 8 IIB Anderson 71 92 13 0 hd Figure 7 7 Project Manager includes the File tree and Statistics table Statistics table displavs MFI and count data for user selected samples 7 8 MasterPlex GT www miraibio com CHAPTER 7 RESULTS TABLES 7 3 Sorting Samples in the Tvping Table The Tvping table displavs the sample data rows in the order that the data were collected in the Luminex system You can sort samples by sample name alpha numeric sort or bv similaritv to the expression level MFI data of a user specified sample Sorting bv Sample Name To sort the Typing table by sample name right click the name that you want to use for the reference and select Sort by Sample gt Ascending or Descending from the shortcut menu that appears Figure 7 8
151. plays the bars in the Multi Compare and Depth bar graphs with a color gradient Displays the MFI and RI thresholds in the Multi Compare bar graphs La Paints only the called alleles in the Multi i Compare and Depth bar graphs Displays the x axis labels vertically in the Multi Compare graph Depth graph and Threshold editing tab MasterPlex GT www miraibio com C 3 APPENDIX C TOOLBARS Table C 3 Multi Graph view toolbar buttons and functions Multi Graph View Toolbar Button Be Vel C 4 MasterPlex GT Function Includes the group and allele name in the x axis label of the Multi Compare graph Depth graph and Threshold editing tab Displays a subset of the x axis labels so that none of the labels overlap in the Multi Compare graph Depth graph and Threshold editing tab Plots the background adjusted MFI data in the Multi Compare and Depth bar graphs Plots the percent relative intensity data in the Multi Compare and Depth bar graphs Puts the Multi Graph view in Two Sample Comparison mode that enables you to plot a Sample by Sample scatter graph Hides or unhides the Heat map in the Multi Graph view Creates a dendrogram of the samples in the active results and displays the Clustering Tool dialog box Displays the Typing table Use the slider to adjust the display angle for the name tags in the Multi Compare graph Displays a drop down list of size options for the horizontal dimension of the bars i
152. ransferred or reexported directly or indirectly into any country prohibited by the Act and the regulations thereunder or will be used for any purpose prohibited by the same GENERAL This agreement will be governed by the laws of the State of California except for that body of law dealing with conflicts of law Future updates of the Software will be available for purchase by licensees for a fee provided a registration card has been received by MiraiBio Inc Should you have any questions concerning this Agreement you may contact Mirai at http www miraibio com You acknowledge that you have read this Agreement understand it and agree to be bound by its terms and conditions You further agree that it is the complete and exclusive statement of the agreement between us iv MasterPlex GT www miraibio com LICENSE AGREEMENT which supersedes any proposal or prior agreement oral or written and any other communications between us in relation to the subject matter of this Agreement MasterPlex GT www miraibio com v LICENSE AGREEMENT vi MasterPlex GT www miraibio com MiraiBio MasterPlex GT 2 0 Genotype analysis software for multiplex data from the Luminex system CHAPTER 1 PAGE Welcome About This Manual LL 1 1 CONTENTS What s New in MasterPlex GT 2 0 1 1 Conventions Used in This Manual 1 1 gereci C UDTUTER aa came ee bb ib eas 1 1 Technical Support 2 2 0 nue enteseng se cuban lt 1 2 CHAPTER
153. rate a report Preview or print a report Save a report 11 1 The Report Manager The Report Manager enables you to select the items you want to include in a report and preview the report 1 To view the Report Manager click the fi toolbar button The Report Manager appears Figure 11 1 It shows the tvpes of information that can be included in a report Report Manager IV Include Sample Information to Report IV Include Cluster Analysis to report J Instrument Status T To add charts to report RIGHT CLICK on M Background Information IV any chart and select Add To Report lV Allele Call Parameter Settings ka A from the Popup Menu f Portrait Landscape e Portrait Landscape Allele Cals a IV Include Allele Call Tables to report Allele Call Table i Allele Frequency Table IV Genotype Frequency Table IV Haplotype Frequency Table Portrait Landscape e Portrait Landscape Vv Paper Size Orientation LETTER Portrait e Portrait C Landscape Preview Report Save Settings As Default Close Figure 11 1 Report Manager summarizes report information 2 To remove an item from the report click the check box to remove the check mark 3 To include raw data MFI percent relative intensity or bead count in the report choose the Include Raw Data to Report option The current view of the Typing table is added to the report MasterPlex GT www miraibio com 11 1 CHAPTER 11 REPORTS 4
154. re provides genotype analysis of results csv from the Luminex system 1 1 About This Manual This manual explains how to use the MasterPlex GT software to import results files csv from the Luminex system set allele calling parameters compute allele genotype or haplotype frequencies sort samples by name expression level or haplotype apply cluster analysis to the MFI data or haplotype generate genotype reports What s New in MasterPlex GT 2 0 New features in MasterPlex GT 2 0 software enable you to perform HLA typing using a lookup table merge results from different bead sets for the same sample allows you to view results from more than 100 different bead sets per sam ple in the Typing table merge results in the Allele Call table automatically launch plug ins when MasterPlex GT starts Conventions Used in This Manual This manual describes the steps required to perform the various tasks associated with the MasterPlex GT software The manual uses a step format to explain the various tasks associated with MasterPlex GT The symbol may follow a step instruction It indicates the software response to the action performed by the user Screen Captures Screen captures may accompany the step instructions for further illustration The screen captures in this manual may not exactly match those displayed on your screen MasterPlex GT www miraibio com 1 1 CHAPTER 1 WELCOME 1 2 Technical Support You can conta
155. rice of the media damaged by accident abuse or misapplication THE ABOVE WARRANTIES ARE EXCLUSIVE AND IN LIEU OF ALL OTHER WARRANTIES WHETHER EXPRESS OR IMPLIED INCLUDING THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE NO ORAL OR WRITTEN INFORMATION OR ADVICE GIVEN BY MIRAI ITS EMPLOYEES DISTRIBUTORS OR AGENTS SHALL INCREASE THE SCOPE OF THE ABOVE WARRANTIES OR CREATE ANY NEW WARRANTIES SOME STATES DO NOT ALLOW THE EXCLUSION OF IMPLIED WARRANTIES SO THE ABOVE EXCLUSION MAY NOT APPLY TO YOU IN THAT EVENT ANY IMPLIED WARRANTIES ARE LIMITED IN DURATION TO NINETY 90 DAYS FROM THE DATE OF DELIVERY OF THE SOFTWARE THIS WARRANTY GIVES YOU SPECIFIC LEGAL RIGHTS YOU MAY HAVE OTHER RIGHTS WHICH VARY FROM STATE TO STATE LIMITATIONS OF REMEDIES Mirai s entire liability to you and your exclusive remedy shall be the replacement of the Software media or the refund of your purchase price as set forth above If Mirai or the Mirai s distributors are unable to deliver replacement media which is free of defects in materials and workmanship you may terminate this Agreement by returning the Software and your money will be refunded REGARDLESS OF WHETHER ANY REMEDY SET FORTH HEREIN FAILS ITS ESSENTIAL PURPOSE IN NO EVENT WILL MIRAI BE LIABLE TO YOU FOR ANY DAMAGES INCLUDING ANY LOST PROFITS LOST DATA OR OTHER INCIDENTAL OR CONSEQUENTIAL DAMAGES ARISING OUT OF THE USE OR INABILITY OF SUCH DAMAGES OR FOR ANY C
156. rinter Name BB HP LaserJet 2100 Series PCL 6 Properties Page range Copies e All Number of copies Current a Pages IV Collate Enter page ee and or page ranges separated by commas For example 1 3 5 12 Print All pages Cancel Figure 11 7 Print dialog box 2 Specify a print range and the number of copies 3 Click OK to print the report 11 3 Opening a Report 1 Open the Report Manager click the ff toolbar button 2 Click the Preview Report The Preview window opens Figure 11 8 11 6 MasterPlex GT www miraibio com CHAPTER 11 REPORTS 72 Preview mE Tux amp Heo rr x 10 21 2002 12 36 32 File Information lt lt C Program Files HitachiSoft MasterPlex GT SampleData Sample1 csv gt gt Program LUMINEX Build VERSION 1 7 69 Date 5 29 02 1 23 45 PM Serial Number LX10000000000 Session 0001 00 0123 Operator SAMPLE Heater Temparature 55 Number Of Samples 72 Minimum Events 0 Background Information A1 beads only Local Background B1 h406 blank Local Background C1 h406 blank Local Background A4 h16236 blank B4 h16236 blank C4 h16236 blank A7 h16401 blank B7 h16401 blank C7 h16401 blank Local Background Local Background Local Background Local Background Local Background Local Background Allele Call Parameters Parameter Set Name Bead Count Threshold 20 Allele Call Parameter Setting Mode Use GroupiLocus Param
157. rsamiiea Projects selected for 7 1 Laversample LayerSamplec layer merge with v LayerSampleG LayerSampleG LayerSample A a LayerSampleT top project LaverSampleA V Delete Project for Merge c LaverSampleC OK Accept Cancel LayerSampleG me f Canai LayerSampleT x If you choose LayerSampleA Delete Project for Merge the Project i Manager shows only the top project name after the merge Figure 4 18 Project Manager 4 18 MasterPlex GT www miraibio com CHAPTER 4 GETTING STARTED 6 Click Accept to merge the projects and keep the Layer Merge wiz ard open Click OK to merge the projects and close the wizard Editing a Bead Name You can edit a bead name in the bead list of the Layer Merge wizard Bead name components include Locus group Allele Suffix The default is no suffix 1 Inthe Layer Merge wizard right click the bead name that you want to edit and select Edit Bead Name from the shortcut menu that appears Figure 4 19 The edit bead name dialog box appears Figure 4 20 FI Layer Merge Wizard Top Project Locus Beads Project Samplet ye T3A Sample 73G Sample B3A Sample IA edt Bead Name 16124C Sample 16126C Sample 161294 Sample Anderson Sample IB 16217C Sample 16223T Sample 16224C Sample Anderson Sample IC1 16292T 16295T Samplel 16294T Sample 16294T 16296T Samplel 16298C Sample 16304C Sample 294T 296T 304C Sample Anderson Sample 74 IC 2 16309G Sample 16
158. rt Items list from the report remove the check mark from the Include Charts to Report option 11 2 Working With a Report in the Preview Window You can preview a report In the Preview window you can print the report save the report open a report perform a text search in the report Previewing a Report 1 To preview a report in the Report Manager click Preview Report Figure 11 3 The Preview window opens and displays the report Figure 11 4 MasterPlex GT www miraibio com 11 3 CHAPTER 11 REPORTS 72 Preview EC jel Fin she AN x 10 21 2002 12 36 32 File Information lt lt C Program Files HitachiSoft MasterPlex GT SampleData Sample1 csv gt gt Program LUMINEX Build VERSION 1 7 69 Date 5 29 02 1 23 45 PM Serial Number LX10000000000 Session 0001 00 0123 Operator SAMPLE Heater Temparature 55 Number Of Samples 72 Minimum Events 0 Background Information A1 beads only Local Background B1 h408 blank Local Background C1 h408 blank Local Background A4 h16236 blank Local Background B4 h16236 blank Local Background C4 h16236 blank Local Background A7 h16401 blank Local Background B7 h16401 blank Local Background C7 h16401 blank Local Background Allele Call Parameters Parameter Set Name Bead Count Threshold 20 Allele Call Parameter Setting Mode Use Group Locus Parameters Prefix Locus Name Ploidy Allele Name RI MFI I I Other 73A 25 0
159. s 8 23 project 4 1 opening 6 15 saving 6 14 Project Manager 3 12 3 13 4 1 4 7 removing projects 4 7 Project Window 3 12 3 13 MasterPlexGT www miraibio com I 2 4 1 4 7 Project window start up window A 2 R relative intensity allele call 6 2 6 3 6 5 requirements hardware 2 1 software 2 1 reset options A 10 results tables Allele Call table 7 11 background color B 5 display defaults B 4 gradient background A 3 A 5 Homology table 7 23 Statistics table 7 7 Typing table 7 1 7 7 S Sample bv Sample scatter graph 8 19 8 21 Sample bv sample scatter graph 7 27 software requirements 2 1 sorting bv expression level Alllele Call table 7 18 Multi graph view 8 3 Typing table 7 9 standards define in lookup table 10 14 10 17 start up window A 2 Statistics table 7 7 T tables see results tables threshold editing 8 16 8 19 toolbar main C 1 Multi Graph C 3 Typing table C 2 e define in lookup table 10 9 10 14 Typing table 4 5 7 1 7 7 bead count view 7 3 7 6 gradient background 7 4 MFI view 7 3 7 5 relative intensitv view 7 3 7 5 sort bv expression level 7 9 striped background 7 4 view graphs for selected samples 7 25 7 27 viewing 7 1 MasterPlex GT www miraibio com I 3
160. s in bitmap format bmp to the a Bitmap system clipboard 8 10 Printing a Graph To print a graph right click the graph and select Print Chart from the shortcut menu that appears 8 11 Adding Graphs to a Report 1 Right click the graph and select Add to Report in the shortcut menu that appears 2 Ifyou want to add all graphs that have been plotted to a report right click the Multi Compare graph and select Add All Charts to Report in the shortcut menu that appears MasterPlex GT www miraibio com 8 23 CHAPTER 8 GRAPHS 8 24 MasterPlex GT www miraibio com CHAPTER CLUSTER ANALYSIS This chapter explains how to apply a cluster analysis to the samples and display a sample dendrogram The MasterPlex GT software can apply cluster analysis to sample genotype or expression data using the following methods Nearest Neighbor Minimum Farthest Neighbor Maximum Median Centroid Between Group Link Ward s Flexible 9 1 Displaying a Dendrogram The Clustering Tool is available in the Typing table or Multi Graph view except for the Allele by Allele tab 1 To display the Multi Graph view of the active results csv or gtp click the Wil button a particular project click Multi Graph under the project of inter est in the Project Manager Figure 9 1 2 To display the Typing table for the active results click the button a particular project click Typing Table under the project of inter est in
161. s td a e Ba beadsnew 7787 Samole Emoty ct ew ast se ee f mu 16224C 22 3 9 7 19 8 23 7 10 1 22 1 21 8 10 9 18 9 23 2 78 94 20 6 e jen 18 8 TK 17 34 a he2 5tz2 Fomm 3 66 7 i e2 e2 as Jsampletmotv 9 8 ASK 1 an 16 64 12 7 15 0 14 6 11 54 15 6 er aa 5063 Jsampletmotv 30 4K 26 1 26 15 12 5 18 74 37 5 Bo k h fe 0 0K OLON 60 0 39 0 7 3 29 3 e Joa ee snore AAS 0 0 20 7 so FFT 98 5 0 04 23 1 86 4 13 64 27 3 pmo as eey SameEmov 27 5 22 54 37 5 A 25 8 280K 28 0 tia 22 ax pa OM ee as 74 17 ak in 7 A lt ii we lt Figure 7 4 Typing table Percent relative intensity data MFI View In this view the Typing table shows the background adjusted MFI of each allele at a particular locus e To display the MFI view click the amp button MasterPlex GT www miraibio com 7 5 CHAPTER 7 RESULTS TABLES The Typing table displays the background adjusted MFI data of each allele Figure 7 5 Note MFI data displayed in red indicates the bead count was less than the user specified event number set in the Parameter Setting dialog box A negative MFI value indicates the background value is greater than the MFI a fe slull ale ee ee ee T O ___ _ ___je __ __ __ __ I eats 2 tpt 24 16126 161298 JAndersorjiezizc J162231 Jis224C Andersorl FR ST ae Sa es PS a a pee sez B4 beadsnew 7787 Jsamp
162. s the sample names and homology score 5 To change the 3 dimensional view of the chart click and hold the mouse while you drag the pointer To reset the view right click the chart and click Reset 3D View from the shortcut menu that appears 6 To copy the Homology chart right click the chart and select Copy As a Bitmap or Copy As Windows Meta Format from the shortcut menu that appears 7 To add the Homology chart to a report right click the chart and select Add To Report from the shortcut menu that appears 7 24 MasterPlex GT www miraibio com CHAPTER 7 RESULTS TABLES 7 6 Viewing Graphs for Selected Samples You can select samples in the Typing table and view the data in the Multi Compare bar graph Depth bar graph or Sample scatter plot See Graphs on page 8 1 for more information Multi Compare and Depth Bar Graph The Multi Compare bar graph displays background adjusted MFI Figure 7 24 The graphs for the selected samples are tiled horizontally to help you compare samples and distinguish differences The Depth bar graph plots the background adjusted MFI or RI for all selected samples in one bar graph Figure 7 25 See page 8 6 and page 8 8 for more information about the Multi Compare and Depth bar graphs 1 Do one of the following to select samples in the Typing table for the graphs To select adjacent samples columns click and hold the mouse while you drag the mouse pointer over the sample names col
163. sen the data are only normalized 1f the standard MFI gt base MFI Set a cutoff MFI value If the standard MFI lt cutoff MFI the data are not normalized SI Lookup Table Editor Export Apply Reset All O4 O8 Oor O ew Locus Setting Name HLA A Version 1 1 Group Name Reset Standard Base MFI Cutoff Enable aoi aoz aos a_o4 aos a stdi JA std2 A_Stdi MI 300 TRUE A Std2 1000 300 TRUE v v Click the Standard Beads button to change the view in the Lookup Table Figure 10 17 Lookup Table Editor Standard Beads view This view shows the standards associated with the alleles of the selected locus 10 14 MasterPlex GT www miraibio com CHAPTER 10 GENOTYPING USING A LOOKUP TABLE 1 Inthe Lookup Table Editor click a tab to select a locus 2 Click the Standard Beads button Cl The Standard Beads view for the selected locus or blood tvpe appears Figure 10 18 a Lookup Table Editor Export Apply Reset All O4 O8 Dor O wo Locus Setting Name Version 10 0 Group Name Reset es Standard Base MFI Cutoff Enable lact lao laos Jao laos JA stdi JA std2 Type Task Standard Beads Figure 10 18 Lookup Table Editor Standard Beads view 3 Click the Add New button EE Alternatively right click the Lookup Table Editor and select Add New from the shortcut menu that appears The New Standard Bead Name Entry
164. splays the allele data using the group color Depth bar graph displays the allele data using the group color Allele Call table displays the group colors 6 8 MasterPlex GT www miraibio com CHAPTER 6 ALLELE CALL PARAMETERS If this option is not chosen the software assigns a different color to each allele You can change the default group or allele color Changing the Group or Allele Color 1 Click the amp button to display the Parameter Setting dialog box Figure 6 7 SI Parameter Setting Group set Cancel OK Save setting as Import Setting Parameter setup for the individual bead Minimum Events 20 count for each bead IV Use group color for Chart and Allele Call Table Lookup Table Allele Name Reportable Level Intensity Threshold Call Intensity G roup SN P2 N 35 selected Group color swatch displays i the color of the selected grou Group Prefix of beads in this group 2 Edit Bead Names g p Group Name IsNP2 TV Change Color Ploidy 1 Diploid C Haploid Other Apply this Ploidy to all groups loci Allele Name H F Group Allele Identifier Allele Call Parameters for SNP2 e Use Relative Intensity for Allele call Reportable Level 25 0 94 of total intensity Intensity Threshold 35 MFI Intensity based Allele Call Call anything bigger than co MFI as an Allele Apply to all groups loci MFI Allele Ratio Figure 6 7 Parameter Setting dialog box Group SNP2 sel
165. sterPlex GT www miraibio com CHAPTER 8 GRAPHS SNPIut 614 SNPI mt 46 mm SNP2ut 732 mm SNP mt 661 SNPS wt 389 SNPS mt 76 TM SNPG ut 522 SNPG mt 78 MM SNP ut 877 SNP mt 60 7 SNPIDUt 653 SNPIO mt 91 Figure 8 12 Multi Compare graph User selected area magnified Moving the Graph You can manually move the graph view in the Project Window 1 Put the mouse pointer over the graph then press and hold the right mouse button 2 To move the graph move the mouse pointer MasterPlex GT www miraibio com 8 15 CHAPTER 8 GRAPHS 8 6 Threshold Editing In the Threshold Editing tab you can view the sample MFI or relative intensity data for a user selected sample Figure 8 13 and Figure 8 14 view the intensity thresholds for the Relative Intensity Allele Call option set in the Parameter Settings dialog box For more infor mation see Relative Intensity Allele Call on page 6 3 change the intensity thresholds 1 To view the intensity data and thresholds click a sample in the H sample list Click the FI toolbar button to view MFI data or the Fl button to view relative intensitv data The Threshold Editing tab displavs a graph of the intensitv data for the selected sample The colored points in the graph represent alleles called in the sample white points represent alleles that were not called 8 16 MasterPlex GT www miraibio com CHAPTER 8 GRAPHS SJS aj7 mf a fee me SJ
166. system clipboard click Copy Info Removing Projects from the Project Manager To remove a project from the Project Manager right click the project name and select Delete from the shortcut menu il NOTE This does not permanentiv delete the file from the svstem MasterPlex GT www miraibio com 4 7 CHAPTER 4 GETTING STARTED 4 2 Opening Luminex Results You can use the menu bar toolbar or the drag and drop method to open Luminex results csv Opening Results Using the Menu Bar or Toolbar 1 Click the Open CSV File button Alternatively select File gt Open CSV File from the menu bar The Open dialog box appears Figure 4 7 Open Look in SampleData fl e Eg Q HLA A_B_DR_BLD csy Sample2 csv LaverSampleA csv 18 sampleBase csv LayerSampleC csv 34 Samplesmall csv LayerSampleG csv LayerSampleT csv Samplel csv Files of type ILuminex Data file csv mdb sl Cancel Figure 4 7 Open dialog box 2 Double click the csv file that you want to open The Project Manager and Project Window appear Figure 4 8 In the Project Manager the file tree displays the file name The Project Window displays the Typing table default 4 8 MasterPlex GT www miraibio com CHAPTER 4 GETTING STARTED BI MasterPlex GT Typing Sample2 Sample2 gtp File Edit View Function Option Window Help O Samplel Typing Table dil Multi Graph tt fe tt tt C Sample2 er
167. tate fe i ha mar sw itabbb fj wee NCE ER RAN PT Typing Table m ee eee ill Multi Graph B laaa lee SamoeEmoy l DO SampleBase Typing Table mo etl es fans reds il Multi Graph Ho fl fale sate O SampleSmall Typing Table jes jew 5012 sana Eres Hi ae 2 isa CS iet dez 5122 Smile Emoty Adjusted MFI Count l Bead ave spo cv a Hs 4a 3q aga Jsampietmatv 14 16124C Mos 102 JI Wt he3 ess Sample Emoty 1A 16126C os 91 H2 i483 5242 Samole Emnty FEET ARET IT 3c saz fa aa e052 jsampietmotv aie s s fameen IB 16223T 53 73 ample Empty Sample Empty IB 16224C 162 11 0 I ON O OC I NNl wee Se Om i n ww OQ mole Empty IB Anderson 38 1 2 A IC1 16292T1 15 41 7 IC1 16294T DO 0 0 IC1 16294T 1 0 6 13 3 y n Ne WO es Se HB WOH ON N oO msm NH NM rm nm ro rl a a al Project Manager Project Window displaying the Typing table includes file tree top and Statistics table bottom Figure 4 8 Project Manager and Project Window NOTE See The Project Manager and Project Window on page 4 1 for more information MasterPlex GT www miraibio com 4 9 CHAPTER 4 GETTING STARTED Opening Results Using the Drag and Drop Method 1 Open Windows Explorer and adjust the window size so that you can view both the MasterPlex GT and Windows Explorer appli cation windows 2 In Windows Explorer navigate to the
168. tensity A 92R7 Y92R7 Other 25 096 35 Amel Amel Other 25 0 35 DYS1 DYS199 Other 25 0 35 DYS3 DYS391 Other 25 0 35 M9 Mg Other 25 0 35 v Group Allele Identifier Group Prefix of beads in this group 2 Edit Bead Names Group Name gaR7 j Ploidy Diploid Haploid Other Apply this Ploidy to all groups loci Allele Name j ju F Applyt Allele Call Parameters for 92R7 e Use Relative Intensity for Allele call Reportable Level 25 0 of total intensity Intensity Threshold 135 MFI C Intensity based Allele Call Call anything bigger than MFI as an Allele Apply to all groups loci MFI Allele Ratio Figure 6 1 Parameter Setting dialog box MasterPlex GT 3 www miraibio com 6 1 CHAPTER 6 ALLELE CALL PARAMETERS Parameter settings and options include Parameter setup for the individual bead Use group color for Chart and Allele Call Table Minimum Events Ploidy Use Relative Intensity RI for Allele Call see See Relative Intensity Allele Call on page 6 3 Intensity Based Allele Call see See Intensity Based Allele Call on page 6 5 Choose this option to apply the parameter settings to a user selected allele bead type If this option is not chosen the group parameters are applied to alleles on a per group basis Choose this option to apply the group color to the allele data in the Allele Call table Multi Compare graph and Depth graph all
169. terPlex GT www miraibio com APPENDIXB PROJECT OPTIONS amp PARAMETERS Allele Call General Options Show Legend Choose this option to display the allele name legend in the Multi Compare graph Show Sample Choose this option to display the allele names in the Labels Tags Multi Compare and Depth bar graphs Figure B 7 Label Tilt Move the slider to the right to rotate the allele name labels counter clockwise in the Multi Compare graph Allele Bar Graph Options These are display options for the bars that represent alleles in the Multi Compare and Depth bar graphs Option View 3D Graph Show Gradation Paint Called Allele Only Graph MFI Value Use Relative Intensity if not checked Show MFI RI threshold lines in bar graphs Vertical Label Thin out labels Show Locus Name and Allele Name Show Heat map Choose this option to Display three dimensional bars in the Multi Compare graph Figure B 7 Display the Multi Compare and Depth graph bars using a color gradient Figure B 8 Apply the group color only to the bars that represent called alleles in the Multi Compare and Depth bar graphs and the graph legends Figure B 9 Plot background adjusted MFI data in the Multi Compare and Depth bar graphs If this option is not chosen the bar graphs plot percent relative intensity Figure B 10 Display the threshold lines in the Multi Compare bar graphs Figure B 7 Display labels vertically along
170. the 8 fe ET gt u Ee Typing table A s jo jap Click a tab to select a locus or C E ft the bloodtvpe a jw uw eo Cl 7 bless l aw T EE e e ki e ms H i i H2777 lidoo1s ess L a oos a oog Click EE to view bv locus GS to show the type to display the Typing table with gradient background an Am number to show ambiguity candidates an Lv number to show inversion candidates Figure 10 2 Steps to type samples using a HLA lookup table 10 2 MasterPlex GT www miraibio com 10i 101 10 101 10 10i 106 10i 106 10 10 10 10i 10 10 CHAPTER 10 GENOTYPING USING A LOOKUP TABLE 10 1 Importing a Lookup Table The import process is carried out only once for each lookup table gtt To import a lookup table 1 Open the MasterPlex GT software 2 In Windows Explorer navigate to the lookup table gtt NOTE Lookup tables for import should be located on the desktop I or a folder other than the Settings folder The software copies the gtt to the Settings folder 3 Double click the file or drag the file to the MasterPlex GT applica tion window Figure 10 3 gt The lookup table is installed copied to the Settings folder and the file name is added to the Lookup Table Selection window Figure 10 5 E MasterPlex GT File Edit View Option Window Help Di om cal ESLA wil zajal alalmi FI MFI Adjusted MFI Count
171. the Luminex results file csv or project gtp of interest 2 Click the Parameter Setting button ah The Parameter Setting dialog box appears Figure 10 7 10 6 MasterPlex GT www miraibio com CHAPTER 10 GENOTYPING USING A LOOKUP TABLE Parameter Setting Group set peers Cancel Save setting as 4 Import Setting T Parameter setup for the individual bead Minimum Events po o count for each bead IV Use group color for Chart and Allele Call Table Lookup Table Frott learner five Lookup Table Allele Name Report Intensity Call Inten A Other HLA A v1 10 Other HLA B v1 10 er E Group Allele Identifier Group Prefix of beads in this group 7 Edit Bead Names Group Name a ns Change Color Ploidy Diploid C Haploid Other Apply this Ploidy to all groups loci Apply to all alleles in the Allele Name Change Color same order in each group Apply to all same name alleles r Allele Call Parameters for A C Use Relative Intensity for Allele call Reportable Level 2s U of total intensity Intensity Threshold 35 MFI e Intensity based Allele Call Call anything bigger than 50 MFI as an Allele Apply to all groups loci Figure 10 7 Parameter Setting dialog box 3 Click Lookup Table The Lookup Table Selection window appears Figure 10 8 MasterPlex GT www miraibio com 10 7 CHAPTER 10 GENOTYPING USING A
172. the Project Manager Figure 9 1 MasterPlex GT www miraibio com 9 1 CHAPTER 9 CLUSTER ANALYSIS D Sample Typing Table ill Multi Graph D Sample2 Typing Table ill Multi Graph File tree D SampleBase Typing Table D SampleSmall Typing Tabl ill Multi Graph MFI Adjusted MFI Count Bead Ave sb cvz EJ 84 22 0 92A7T 193185 7 96 3 me 216 248 8 115 1 me 144 164 0 114 1 YS199C 228 257 9 113 2 Y51997 24 224 91 8 IM YS391C 247 279 5 113 4 Statistics table 53916 67 69 5 103 6 IC 51 20 2 39 3 100 77 4 77 2 RY 4e5C 144 154 1 107 2 RY 465T 37 30 3 81 4 RY 15324 7 64 98 5 AY1532G 80 91 8 114 7 RY 3225C 286 326 3 114 0 RY 3225T 55 58 6 107 0 141 150 1 106 5 FI Figure 9 1 Project Manager 3 Click the Show Dendrogram toolbar button aF The Clustering Tool window Figure 9 2 and the dendrogram Figure 9 3 and Figure 9 4 are displayed The Wards method and cluster by genotype are the defaults The Genotype option clusters samples according to the genotype called for the alleles The Expression option clusters samples according to the MFI data for the alleles ii NOTE The Show Dendrogram button 2 is not available in the Allele bv Allele tab SampleSmall Clustering Tool fx ALA Min FON Max Median Centroid B G Link Ward s Flexible f Genotype C Expression Figure 9 2 Clustering Tool window 9 2 MasterPlex GT www miraibio com
173. the x axis of the graphs Show a subset of the labels along the x axis of the graph so that none of the labels overlap Show both the locus group and allele name on the x axis of the graph Display the Heat map in the Multi Graph view MasterPlex GT www miraibio com B 7 APPENDIX B PROJECT OPTIONS amp PARAMETERS Adjusted MFI of 1 B14 PUNDJBXIER 4py SMPS SMP SMPS SMP4 SNPS SNIPE SMPF SMPS SHP2 wake Adjusted MFI of 1 B1 100 threshold D mW Oo WW 0 Wo Wi ot PUNDJOJIEB L A SMFPE SMPs G F4 SMPs SHIP a5 a0 25 20 SMPS SMP SMPS SMP SMP Figure B 7 Multi Compare graph 3D bars top 2D bars bottom www miraibio com MasterPlex GT B 8 APPENDIXB PROJECT OPTIONS amp PARAMETERS Adjusted MFI of 1 B13 Mk i xi L EEA rere LI I I LI 1 LI I LI 1 LI I I 1 acco 1 1 l TIT atti 1 wus mt PUACIE YER L I SHIPS SHFPIO SHP2 SHPS SHP4 SNPS SMP6 SH P7 SNPS SHF Adjusted MFI of 1 51 Mb LI SMFS A i 7 1 w SHPO SHFIO SMFS SMPY SNPS wits At SNP4 1 seo eoho food SHP2 SMFS SNFI puno yeg JI Figure B 8 Multi Compare graph solid color bars top gradient color bars bottom www miraibio com B 9 MasterPlex GT APPENDIX B PROJECT OPTIONS amp PARAMETERS Adjusted MFI of 1 B1 BUROJBXIEB JIN SNPS SMP SNPS SHMP4
174. tions Selecting a Reference Sample You can specify a reference sample in the Allele Call table Two viewing options are available you can paint highlight the alleles that are called the same as the reference sample or you can paint the alleles that are called different from the reference sample 1 Inthe Allele Call table make sure the Reference Sample Selection radio buttons are displayed Figure 7 14 If the radio buttons are not displayed click the lill button to displav them Sample 6 selected for the reference BI Allele Call SampleSmall SampleSmall gtp SNP1 SNP2 SNP3 SNP4 SNP5 SNP6 JSNP7 SNP8 SNPS SNP10 G1 6 Hi 7 r B2 9 C iaz Fi 5 FOIBIJ1 FIBSJIO r E1 4 r cij2 r D113 C A1 no dna v wewt wewt wewt wewt wewe wewe wewt wewt wh wk wk wt wewe owkwk wwe wkwk wkwk wewe wwe wkwk wkwk wE wt wk wl wewt wewt wk fe wewt wewe wewt wewe wwe wt wt wE wE wkwk wwe wkwk wkwk wewe owhwk wewt wkwk wE wE tib ab wkwk wkwk wkwk wkwk owhwk wewt wet wewe tib wE wE wE wkwk wwe wkwk wkwk wewe wE jab wewt wwe wE wt Wewe wewe wewt we ME wewe wewt wewe wewt wewe wewt wewe wewe wewt wewt meme memet wewe wewt wewe wE wt wk wk wE ME wewt wewt wewt wewe wewt wewe wewt wt wt wk wt wt mME mtmt wE meme omt mt wewt wewe wewe wE wt dd i XI U SI XI gt Figure 7 14 Allele Call table Alleles called different from the reference sample 6 are painted 2 Click t
175. tions available to you and the user modifiable parameters for new projects To view the project options and parameters 1 Select Option 7 Set New Project Default Parameters from the menu bar The New Project Default Parameters dialog box opens Figure B 1 Allele Call General Options Parameter setup for the individual bead IV Use group color for Chart and Allele Call Table Minimum Events 20 count for each bead Ploidy Diploid Haploid Other Allele Call Parameters e Use Relative Intensity for Allele call Reportable Level 25 of total intensity Intensity Threshold 35 MFI C Intensity based Allele Call Call anything bigger than 50 MFI as an Allele NOTE These parameters are effective only to new projects OK Reset All Cancel Figure B 1 New Project Default Parameters dialog box Allele Call Parameters tab MasterPlex GT www miraibio com B 1 APPENDIX B PROJECT OPTIONS amp PARAMETERS B 1 Allele Call Parameters In the Allele Call Parameters tab Figure B 1 you can specify defaults for the Parameter Settings dialog box Figure B 2 Allele Call General Options Parameter setup for Choose this option to specify allele call parameters the individual bead for an individual bead type Use group color for Choose this option to display the group colors Chart and Allele specified in the Parameter Settings dialog box in Call table the Allele Call table Multi Comp
176. tom Colors The color palette shows the custom color options Figure 10 16 Luminosity slider Custom color field Color swatch ue 105 Red 85 Sat 186 Green 234 ColorlSolid Lum f150 Blue 179 Cancel Add to Custom Colors Figure 10 16 Color palette Custom color options d Use the click and drag operation to move the cross hairs in the custom color field Adjust the color brightness using the lumi nosity slider The Color swatch shows the color selection MasterPlex GT www miraibio com 10 13 CHAPTER 10 GENOTYPING USING A LOOKUP TABLE 10 Tih 12 13 14 e When you are finished defining the color click Add to Custom Colors to apply the color and click OK Enter the genotype frequency Click the alleles and standards that you want to include in the gen otype expression pattern To define another allele expression pattern for the selected locus follow step 4 to step 10 To define the allele expression patterns for another locus or the blood type follow step 1 to step 11 To copy the Click Apply when you finish defining the genotype expression pat terns Setting Standards In the Standard Beads view of the Lookup Table Editor Figure 10 17 you can Associate alleles or a blood group with a standard Choose normalization for a standard and the associated allele MFI data Set a base MFI threshold for a standard MFI When the normaliza tion option is cho
177. tomatic Background Sample Detection on data load 4 To define a keyword click Add New Keyword enter the keyword in the dialog box that appears and click OK Figure 5 3 The keyword is added to the Background Keywords list Figure 5 2 NOTE A keyword added during a session is applied only to subsequently opened results Figure 5 3 Add a Background Keyword dialog box 5 4 MasterPlex GT www miraibio com CHAPTER 5 NEGATIVE CONTROLS 5 To delete a keyword select the keyword you want to delete in the Background Keywords list and click Delete Selected Keyword At the prompt click Yes MasterPlex GT www miraibio com 5 5 CHAPTER 5 NEGATIVE CONTROLS 5 6 MasterPlex GT www miraibio com CHAPTER ALLELE CALL PARAMETERS The MasterPlex GT software can use median fluorescence intensity MFI or relative intensity RI data to call alleles This chapter explains how to set parameters for the allele calling algorithm 6 1 Parameter Settings and Options To display the parameter settings click the Parameter Setting but ton fab The Parameter Setting dialog box appears Figure 6 1 Parameter Setting SEs Group set xl Cancel OK Save setting as Import Setting F Parameter setup for the individual bead Minimum Events 20 count for each bead Use group color for Chart and Allele Call Table Lookup Table Lookup Table Allele Name Reportable Level Intensity Threshold Call In
178. tton right click a chart graph and select Add to Report from the pop up menu that appears The graph name is added to the chart items list in the Report Manager and the Chart Preview window displays a thumbnail of the chart Figure 11 3 11 2 MasterPlex GT www miraibio com CHAPTER 11 REPORTS Report Manager Sample by Sample Scatter h408 1 vs h16236 Allele by Allele Scatter I 16126C vs IB 1622 I Include Sample Information to Report C h a rt Instrument Status a V Background Information Ite ms ist iv Allele Call Parameter Settings e Portrait Landscape e Portrait Landscape IV Include Allele Call Tables to report P Include Raw Data to report WV Allele Call Table IV iv Allele Frequency Table fu IV Genotype Frequency Table r V Haplotype Frequency Table lal eview Windo e Portrait C Landscape e Portrait Landscape Chart thumbnail paz p his in the iv Include Charts to report Paper Size Orientation LETTER Portrait e Portrait Landscape Chart F review en Preview Report Save Settings As Default Close window Figure 11 3 Report Manager 2 To add all charts to the report right click a chart and select Add All Charts to Report from the pop up menu that appears All of the chart names are added to the Chart Items list 3 To change the display in the Chart Preview window click the chart of interest in the Chart Items list 4 To exclude the graphs in the Cha
179. tv 1 2 1 ka l a 1 2 2 D2 a3 37 JSampleEmotv 1 1 2 1 4 3 5 2 Dba a a 8952S le Emoty a 2 2 11 4 3 3 1 Hoo o ea ess Sample Emoty 2 z 1 8 2 1 4 3 H2 ea 5242 SamoeEmty 1 z 1 2 3 4 3 3 H3700 48340000 49728 JsampieEmetv 1 z 1 2 4 1 3 1 lt gt ff Blue color gradients Red color gradients shows relative shows relative expression levels of expression levels of alleles at the first alleles at the next locus IA locus IB Figure 7 3 Typing table Gradient background indicates relative expression levels of the alleles at a locus 7 4 MasterPlex GT www miraibio com CHAPTER 7 RESULTS TABLES Relative Intensity View In this view the Typing table displays the percent of the total intensity RI for each allele at a particular locus In a biallelic analysis the relative intensity of allele a and allele b is computed as follows RI MFL MFI MFI x 100 RI MFI MFI MFI x 100 e To display the relative intensity view click the fo button The Typing table displays percent relative intensity for each allele represented in the bead set Figure 7 4 NOTE RI data displayed in red indicates the bead count was less than the user specified event number set in the Parameter Setting dialog box l i 0 l ti am ofr 2 sal 80 pI eS ae ES ee JB p l o eas JiGr24C Jini2eC 161294 andersor 16217 _ 16223T_ WellName Samole Name TotaiEvents noes J d TT Li el tnin f3 Ja
180. umn headers Click the mouse when you complete the selection Alter natively press and hold the Shift key while you click the first and last sample name in the selection To select nonadjacent samples press and hold the Ctrl key while you click the sample names 2 Right click a selected sample name and select Open Bar Graph from the shortcut menu that appears The Multi Graph view appears Figure 7 24 3 Click the Multi Compare tab to view a separate bar graph of MFI or RI data for each selected sample Figure 7 24 4 Click the Depth tab to display the Depth bar graph Figure 7 25 5 To return to the Typing table for the active results click the button MasterPlex GT www miraibio com 7 25 CHAPTER 7 RESULTS TABLES Adjusted MFI of h16236 5 H4 nono om am an enn pm nm mn rrr npr rrr rrr nq A 5 200 ie a en eee ene Se an 5150 41 z i R p x lee mm ew ada aw eewteaeweww eae wo lew ees we dewe ow wo ew Lewww ene wee m 100 1 1 1 1 i l 1 D eoep 1 es Ta see ee ae aes Pee MT Ue patina L sff i LE a G A EL SATE EL OE AAT LOS 734 14 16126C IC1 162941 C2163194 IIB146C IIB152C IIC 199C Adjusted MFI of h16236 14 46 T 200 ee Reet ee es Ae Oe 5150 R4100 0 WO hails ei ET aioe ice e Ria sie O GA eS Se eee a lt 50 E iene GP COLL LA Me MA AE COIS EAS SEB 734 14 16126C IC1 162941 C2163194 IB146C IIB152C IIC 199C Adjusted MFI of h16236 6 45
181. user selected sample is displayed at the top of the sample list and the remaining samples are sorted by similar expression level descending order Resetting the Sort To reset the sample list to the default sort the order that the data were collected by the Luminex system right click the sample list and click Reset Sample Sorting from the shortcut menu that appears Cis This also resets the sample sort in the Typing table MasterPlex GT www miraibio com 8 3 CHAPTER 8 GRAPHS 8 2 Heat Map The Heat map is a color coded representation of background adjusted MFI data for each sample The color range from black low to red high represents the allele expression level The Heat map provides a convenient way to quickly compare the expression level of the alleles in a single sample as well as the expression level of a single allele across multiple samples The map rows organize the alleles from left to right in the same order as the Statistics table from top to bottom Figure 8 3 A map represents one sample and shows the expression level of each allele in the sample Each column in the map represents one allele and shows the expression level for that allele across all of the samples el ofa ml ji 8 fer LI Samplel Typing Table Pos SampeName JI il Multi Graph LI Sample2 Typing Table il Multi Graph LI SampleBase Typing Table O ai 1 SNP3 wt MFI 953 Typing Table 91 5 E dl Multi Graph MFI Adjusted
182. ware can use a lookup table gtt to call genotypes As an example this chapter explains the steps to HLA typing using a lookup table and how to import or setup and manage lookup tables Figure 10 1 and Figure 10 2 provide an overview of the steps to perform HLA typing in MasterPlex GT using a lookup table 1 Open Luminex results csv 8 fo H u ale a ESS aan eee a ae A A fu fos jo iI Sta2 WellName Sample Name TotaEvents Notes on A JI f f ee pa FT Bi aom sess o et fiaoooa asso pr faooos fasa E1 faooos faso S fi faooos cots ei ifiaooog fass 17 wi faoooz seo S a2 _ifiaooos_ fass o 2 faooogs fsa o c2 fomo sor p2 faon fas o E2 loma Jssis J F2 foma seg f 62777 faoms fsoss h2 loms less d 268 268 268 18 18 268 268 268 68 18 268 268 268 268 268 MasterPlex GT Typing HLA A_B_DR_BLD 0 2 Im po rt or create a File Edit View Function ion Window Help Option HLA looku table tt Di ma ele da sala lalm To im T M Dances fi jebiji ma tal k p 1 dl Multi Graph double click the gtt or drag it to the MasterPlex GT application window MFI Adjusted MFI Count im Settings File Edit view Favorites Tools Help Q sxx 7 Q Search gt Folders E Address Name Size Date Modified B C Program F

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