Mentype ® Chimera®
Contents
1. Figure 9 e iz OE zes iz Oz 5 295 2 ze 92 6b BED e Z L8 Es tz Z bz 28 Z OV a de se Ee 2 06 2 zz 92 2 22 02 8 94 lt zt ou E 2 gH EH HH 5 E 82 92 Ize 287 292 z 22 Lz er ekler Eri 6 PE 9 Ei Ob tk ZH oV 6 8 La 9 L a Be TE ag a 5 8 oS 2 0002 co E 5 5 2 m 007 ooe 002 00 Eo ETS 9202501 etw o eo E ER ae 772 ran zw zz m 2 92 26 02 ez 92 fez HOZ V 2b 82 92 fez 22 oz Sb ev e zv 0 84 94 e 2 Ou 9z tz zz 02 84 55 a za ss jaza jr ez rez sa Ii zz kaj ren zz ez Ez rz en zen ea z e e a 2808919 lt NNNM poe evan 1 ee Y ot 2 0001 E Os e o 0002 BI SO oor 008 005 00 ES ga 99025124 195811 00925 2011581 Bo SB r Et fa s s fek 24 84 fa zz 2 lez pz zz 08 Ey e oz Ex 9 Ey 2 sz Ez Fz 59 av n 8 amp SH HM 16 EDI EWI 182 2 92 22 ej BV sz ez2. enne 3 1 Matrix generation esee 3 2 Sample preparation 3 3 Setting up the GeneScan software Analysis DaralTietor evan Electrophoresis using the ABI PRISM 3100 Avant 3100 Genetic Analyzer 16 4 1 Spectral calibration matrix generation 4 2 Sample preparation 4 3 Setting up the GeneScan software e 4 4 Analysis parameter sse Electrophoresis using the ABI PRISM 3130 3130xl Genetic Analyzer 5 1 Spectral calibration matrix generation e UEE UUUEUjUj 21 5 2 Sample preparatin sene oniinn i nan 24 5 3 Setting up the GeneMapper ID software re 25 5 4 Analysis parameter analysis method seeene 27 Electrophoresis using the ABI PRISM 3500 3500xL Genetic Analyzer 28 6 1 Spectral calibration matrix generation 6 2 Sample preparation 6 3 Setting up a run 7 1 Biotype template files Y 2 CODlTOIS us dca tre mre 7 3 Lengths of fragments and alleles Interpretation of results Population genetic data 11 Explanation of Symbols sse Mentype Chimera December 2014 LE
3. Bio Es eT o Diagnostic GmbH Mentype Chimera Manual The new standard for chimerism analysis In Vitro Diagnostics 2 100 400 1000 Li Version January 2013 45 13210 0025 45 13210 0100 45 13210 0400 45 13210 1000 LOT Batch Code Biotype Diagnostic GmbhH u Moritzburger Weg 67 D 01109 Dresden Germany Made in Germany Biotype Diagnostic GmbH develops produces and markets their PCR based rapid Mentype Detection Kits Our products provide customers with fast and reliable testing methods for professional medical diagnostics Our Mentype Test Kits guarantee highest quality standards for clinical research and diagnostics For information and enquiries about the Mentype Chimera PCR Amplification Kit please do not hesitate to get in touch or visit www biotype de en home html Mentype Chimera December 2014 LEUGACHNv1en Mentype Chimera Product description Mentype Chimera is a multiplex PCR application specifically developed for chimerism monitoring after blood stem cell and bone marrow transplantation respectively The assay was validated by chimerism analysis of over 200 HLA matched related donor recipient pairs and its suitability was confirmed in a comparative clinical evaluation study Ever since the assays is successfully used in routine diagnostics Genetic markers that are addressed by Mentype Chimera are distributed over 12 chromosomes and represent highly polymorphic
4. 10 mM Tris HCI pH 8 0 and 1 mM EDTA e g 0 1 x TE buffer The primer mixes are adjusted for balanced peak heights at 30 PCR cycles and 0 5 ng Control DNA XY5 in a reaction volume of 25 ul If more DNA template is applied higher peaks can be expected for small PCR fragments and relatively low peaks for large fragments Reduce the amount of DNA template to correct this imbalance Positive control For the positive amplification control dilute Control DNA XY5 to 0 5 ng ul Instead of template DNA pipette diluted Control DNA into a reaction tube containing the PCR master mix Negative control For the negative amplification control pipette nuclease free water instead of template DNA into a reaction tube that contains the PCR master mix Template DNA Sometimes measured DNA concentration varies depending on the quantification method used It might thus be necessary to adjust the optimal DNA amount Mentype Chimera December 2014 LEUGACHNv1en 2 2 PCR amplification parameter Perform a hot start PCR in order to activate the Multi Tag2 DNA Polymerase and to prevent formation of non specific amplification products Number of PCR cycles depend on the amount of DNA applied 30 PCR cycles are recommended for all samples In case of critical samples 100 pg DNA the number of PCR cycles can be increase from 30 to 32 Standard method Recommended for all DNA samples Temperature Time 94 C 4 min hot start for activat
5. 7 2 Gontrols The Control DNA XY5 of the test kit and other commercially available DNA from standard cell lines represent the following alleles Table 3 Allele assignment of Mentype Chimera i Control ATCC CCR CCR CCR mous DNA XY5 K 562 9947A 9948 3657 Amelogenin XY X X X X XN XN 0251360 22 25 20 28 23 24 22 25 22 23 0351744 17 18 18 18 1717 18 18 14 17 D4S2366 9 12 13 13 11 13 9 14 9 14 0552500 10 11 15 15 15 16 11 15 11 16 D6S474 15 16 14 17 13 17 16 16 15 16 0751517 22 27 21 24 25 19 25 20 22 24 25 0851132 18 20 20 24 19 21 20 24 1718 01052325 1314 7 13 9 10 8 14 9 14 0125391 17 19 23 23 18 20 18 24 18 19 018551 13 15 15 16 15 19 15 18 12 20 02152055 25 27 28 35 19 1 26 19 1 26 19 1 25 SE33 15 21 2 26 2 28 2 19 29 2 23 2 26 2 22 2 27 2 For further confirmation the table above displays alleles of reference DNA purchased from ATCC http atcc org Produtcs PurifiedDNA cfm celllines as well as three assignments of reference DNA purchased from Coriell Cell Repositories CCR http locus umdnj edu nigms standard of Szibor et al 2003 7 3 Lengths of fragments and alleles Table 4 to table 6 show the fragment lengths of individual alleles that refer to the DNA Size Standard 550 BTO All analyses have been performed on an ABI PRISM 310 3130 Genetic Analyzer with POP 49 polymer Different analysis instruments DNA size standards or polymers may result in different fragment lengths In addition a visual alignment with the
6. 4 capillaries is named ABI 3130 and the system with 16 capillaries is named ABI 3130xl The virtual filter set AnybDye shall be used for the combined application of the five fluorescent labels 6 FAM BTG BTY BTR and BTO the matrix standard will be called BT5 hereinafter Material Capillary 36 cm Capillary Array for 3130 3130xl Polymer POP 4 Polymer for 3130 Buffer 10x Genetic Analyzer Buffer with EDTA 5 1 Spectral calibration matrix generation Prior to conducting DNA fragment size analysis it is necessary to perform a spectral calibration with the fluorescent labels 6 FAM BTG BTY BTR and BTO for each analyzer The calibration procedure creates a matrix that is used to correct the overlap of fluorescence emission spectra of the dyes Spectral calibration comprises the following steps Preparation of spectral calibration standards Loading standards to the 96 well reaction plate one sample per capillary Creating the instrument protocol for spectral calibration Protocol Manager Defining the plate composition in the plate editor Plate Manager Performing a spectral calibration run and checking the matrix Mentype Chimera December 2014 LEUGACHNv1en 22 Setting up the spectral calibration standards Example for 4 capillaries ABI 3130 Component Volume Hi DiTM Formamide 60 0 ul Matrix standard BT5 5 0 yl Load 12 ul of the mix to a 96 well reaction plate e g position A1 D1 Denaturation for 3
7. Error Status in the Event Log or examine the quality of the raw data for each capillary in the Capillaries Viewer or the Cap Array Viewer View data as overview in Run History or Cap Array Viewer of the Data Collection Software Run data are saved in the Run Folder of the previously chosen Result Group Mentype Chimera December 2014 LEUGACHNv1en 27 5 4 Analysis parameter analysis method The recommended analysis parameters are Peak Detection Algorithm Advanced Ranges Analysis Partial Range Start Pt 2000 Stop Pt 10000 Sizing All Sizes Smoothing and Baselining Smoothing Light Baseline Window 51 pts Size Calling Method Local Southern Method Peak Detection Peak Amplitude Thresholds BY G R 0 Min Peak Half Width 2 pts Polynominal Degree 3 Peak Window Size 11 pts Slope Thresholds 0 0 The peak amplitude threshold cut off value corresponds to the minimum peak height that will be detected by the GeneMapper ID ID X software The thresholds are usually 50 200 RFU and should be determined individually by the laboratory Recommendation The minimal peak height should be three times as high as the background noise of the baseline Point alleles i e alleles with at least 1 bp difference to the next integer allele may occasionally not be distinguished For improved peak detection minimise the Peak Window Size further Mentype Chimera December 2014 LEUGACHNv1en 28 6 Electrophoresis
8. allele Size bp 0851132 BTG 018551 02152055 BTG 12 1 17 12 13 8 24 7 16 351 13 1 21 9 245 9 2 17 355 14 1 25 14 3 0 249 18 359 15 28 10 2 25 19 363 16 32 1 053 112 20 367 17 36 2 257 122 21 371 18 40 3 26 13 2 22 375 22 19 44 4 264 142 23 3 8 231 20 48 5 268 24 382 21 51 6 072 162 25 386 22 55 7 276 26 390 23 59 17 2 278 173 27 395 24 63 8 279 28 399 25 67 18 2 281 29 403 26 71 9 283 192 30 406 27 75 20 287 31 411 21 291 32 415 D5S2500 BTG 212 293 33 419 9 88 22 295 34 423 0 92 23 299 231 35 427 1 96 24 302 36 431 2 200 25 306 37 435 38 3 204 26 310 39 443 4 208 27 314 5 212 28 318 29 6 216 7 220 8 224 Mentype Chimera January 2013 41 42 Table 6 Fragment lengths of the Mentype Chimera allelic ladder analysed on an ABI PRISM 3130 Genetic Analyzer with POP 49 polymer yellow panel Marker allele Size Marker allele Size bp Marker allele Size bp en 01052325 BTY SE33 BTY SE33 BTY 6 21 6 3 205 4 2 5 8 25 2 278 7 26 7 3 209 7 26 2 282 26 8 31 8 210 8 2 27 2 285 27 9 36 9 214 9 2 28 2 289 28 28 3 10 41 0 218 29 2 293 29 11 45 10 2 220 30 2 297 30 12 50 1 222 112 31 2 301 31 13 55 2 226 12 2 32 303 14 60 3 230 32 2 305 15 65 13 2 232 13 3 33 307 16 70 4 234 14 2 14 3 33 2 309 17 75 18 5 238 34 311 19 85 15 2 240 34 2 313 16 241 16 2 16 3 35 315 7 245 17 2 17 3 35 2 317 8 249 36 318 18 2 251 18 3 36 2 321 9 253 37 322 37 2 19 2 255 38 326 39 42 20 257 20 1 49 3
9. gt Add Sample to Project open folder of current run Select a matrix sample in the Sample File column Sample Raw Data Check the matrix samples for a flat baseline As shown in the figure below there should be at least five peaks with peak heights about 1000 4000 RFU Y axis for each matrix sample optimal range 2000 4000 RFU 5 Tura wal HAT AT 77 gt rx io o gt gt xx Vv 2900 Data Points X Fig 1 Electropherogram with raw data of the matrix standard 6 FAM Select an analysis range with flat baseline and re inject the matrix sample if necessary Note down start and end value data points of the analysis range e g start value 2900 end value 5400 Calculate the difference e g 5400 2900 2500 data points Mentype Chimera December 2014 LEUGACHNv1en 12 Generation of a new matrix Tools GeneMapper Manager Matrices gt New Create the matrix name e g Matrix BT5 Import matrix samples for all dyes B G Y R O Click on the symbol Matrix Editor Matrix Description Matrix Name Security Group Y SOUP GeneMapper ID X Security Group iv Description p Matrix Settings Select the Matrix Standard Sample File Number of Dyes 5 No File Selected for B Data Start At 1000 No File Selected for G Data Start At 1000 No File Selected for Y Data Start At 1000 No File Selected f
10. more matrix standard for spectral calibration Check the new matrix with your current samples There should be no pull up peaks between the dye panels B G Y R O with the new matrix 5 2 Sample preparation Component Volume Hi Di Formamide 12 0 ul DNA Size Standard 550 BTO 0 5 yl prepare 12 ul of the mix formamide DNA size standard for all samples add 1 ul PCR product diluted if necessary or allelic ladder Denaturation for 3 min at 95 C Cool down to 4 C and place samples on the autosampler tray Since injections take place simultaneously on all capillaries 4 or 16 samples must be pipetted on the plate of multi capillary analyzers If fewer samples are analysed the empty positions must be filled with 12 ul Hi DiTM Formamide To ensure a reliable allelic assignment on multi capillary analyzers several ladders should be run Room temperature may influence the performance of PCR products on multi capillary instruments so that shoulder peaks or split peaks occur especially at low temperatures Pay attention to keeping ambient conditions as recommended by the instrument manufacturer Optimal will be a stable room temperature gt 22 C Signal intensities Options to increase the signal intensity Reduce the volume of the DNA Size Standard 550 BTO to peak heights of about 500 relative fluorescent units RFU Purify the PCR products before starting the analysis Mentype Chimera December 2014 L
11. using the ABI PRISM 3500 3500xL Genetic Analyzer For detailed instructions on instrument setup spectral calibration or application of the Applied Biosystems 3500 Series Data Collection Software version 1 0 and the GeneMapper ID X software version 1 2 refer to the Applied Biosystems 3500 3500xL Genetic Analyzers User Guide The system with 8 capillaries is named AB 3500 and the system with 24 capillaries is named AB 3500xL The virtual filter set AnybDye shall be used for the combined application of five fluorescent labels 6 FAM BTG BTY BTR and BTO the matrix standard will be called BT5 hereinafter Material Capillary 36 cm Capillary Array for 3500 3500xL Polymer POP 4 Polymer for 3500 3500xL Buffer 10x Genetic Analyzer Buffer with EDTA for 3500 3500xL 6 1 Spectral calibration matrix generation Prior to conducting DNA fragment size analysis it is necessary to perform a spectral calibration with the fluorescent labels 6 FAM BTG BTY BTR and BTO for each analyzer The calibration procedure creates a matrix that is used to correct the overlap of fluorescence emission spectra of the dyes Spectral calibration comprises the following steps Preparation of spectral calibration standards Loading the standards to the multi well reaction plate one sample per capillary Preparation of instrument and creating a Dye Set BT5 Performing a spectral calibration run and checking the matrix Mentype Chimera December 2014
12. 061 7 0 097 20 0 101 4 0 042 8 0 005 21 0 099 5 0 213 22 0 082 6 0 103 PIC 0 740 23 0 077 T 0 009 PD 0 918 24 0 155 8 0 007 HET 0 733 25 0 230 26 0 054 PIC 0 780 27 0 014 PD 0 938 28 0 005 HET 0 804 PIC 0 860 PD 0 967 HET 0 826 Tabelle 9 Population genetic data Marker 0851132 Marker D10S2325 Marker 0125391 Allele Allele frequency Allele Allele frequency Allele Allele frequency 16 0 007 6 0 002 15 0 035 17 0 095 T 0 102 16 0 019 18 0 221 8 0 056 17 0 107 19 0 153 9 0 121 17 3 0 019 20 0 128 0 0 142 18 0 215 21 0 119 1 0 144 18 3 0 007 22 0 133 2 0 193 19 0 121 23 0 077 3 0 133 19 3 0 016 24 0 056 4 0 065 20 0 117 25 0 005 5 0 037 21 0 093 26 0 005 6 0 005 22 0 114 27 0 002 23 0 072 PIC 0 860 24 0 040 PIC 0 850 PD 0 967 25 0 021 PD 0 964 HET 0 851 26 0 002 HET 0 828 PIC 0 870 PD 0 971 HET 0 893 Mentype Chimera January 2013 46 Tabelle 10 Population genetic data Marker D18S51 Marker 02152055 Marker SE33 ACTBP2 Allele Allele frequency Allele Allele frequency Allele Allele frequency 10 0 005 16 1 0 056 11 0 002 12 0 103 7 1 0 021 12 0 014 13 0 110 18 1 0 023 13 0 002 14 0 157 19 1 0 274 13 2 0 002 15 0 199 20 1 0 040 14 0 026 16 0 161 21 1 0 019 15 0 049 17 0 112 22 1 0 005 16 0 047 18 0 072 23 0 007 TE 0 070 19 0 028 25 0 112 17 3 0 002 20 0 030 26 0 116 18 0 044 21 0 021 27 0 016 18 3 0 002 24 0 002 28 0 007 19 0 082 29 0 030 19 2 0 009 PIC 0 850 30 0 021 20 0 044 PD 0 964 31 0 023 20 2 0 009 HET 0
13. 69 50 20 2 259 21 261 21 2 263 22 22 2 267 23 2 270 23 24 2 274 24 25 276 rounded to integer The off ladder alleles of Biotype s DNA pool are allocated with the actual Biotype template files for GeneMapper ID or Genotyper software For further alleles see amongst others http www cstl nist gov biotech strbase str_fact htm t For better orientation these alleles are heightened within the allelic ladder Mentype Chimera January 2013 43 8 Interpretation of results As mentioned above post PCR analysis and automatic allele assignment with suitable analysis software ensure a precise and reliable discrimination of alleles An automated calculation of the donor recipient DNA ratio as well as standard deviations and detection limits can be obtained directly from raw data of a fragment size analysis using e g Chimeris Monitor Software from Biotype Diagnostic GmbH If results that are obtained with Mentype Chimera should be harmonized to results from cytological analyses make sure that cytological analyses were performed with at least 500 leucocytes Pull up peaks Pull up peaks may occur if peak heights are outside the linear detection range or if an incorrect matrix was applied They appear at positions of specific peaks in other color channels typically with lower signal intensities Stutter peaks The occurrence of stutter peaks depends on the sequence of the repeat structure and the number of alle
14. 902 32 0 026 21 0 035 33 0 067 212 0 019 34 0 074 22 0 007 35 0 053 22 2 0 035 36 0 007 23 2 0 023 97 0 002 24 0 002 24 2 0 035 PIC 0 870 25 2 0 044 PD 0 971 26 2 0 040 HET 0 856 27 2 0 084 28 2 0 084 29 2 0 051 30 0 002 30 2 0 061 31 2 0 028 32 2 0 023 33 0 009 33 2 0 005 34 0 002 36 0 002 PIC 0 950 PD 0 990 HET 0 949 All population genetic data based on an analysis of ca 210 unlinked Caucasians performed by Biotype Diagnostic GmbH Mentype Chimera January 2013 47 10 References B r W Brinkmann B Budowle B Carracedo A Gill P Lincoln P Mayr W Olaisen B 1997 DNA Recommendations Further report of the DNA commission of the ISFG regarding the use of short tandem repeat systems nt J Legal Med 110 175 176 Becker D Vogelsang D Brabetz W 2007 Population data on the seven short tandem repeat loci 0452366 065474 0145608 0195246 0205480 0215226 and 0225689 in a German population nt J Legal Med 121 78 81 Botstein D White RI Skolnick M Davis RW 1980 Construction of a genetic linkage map in man using restriction fragment length polymorphisms Am J Hum Genet 32 314 331 Hering S M ller E 2001 New allele and mutational events in D12S391 and 0851132 sequence data from an eastern German population Forensic Sci Int 124 187 191 Jones DA 1972 Blood samples Probability of Discrimination J Forensic Sci Soc 12 355 359 Nei M Roychoudhury AK 1974 Sampling variances of heterozygosity and g
15. Biotype templates for GeneMapper ID ID X Software are Panels Chimera Panels v1 v1X or higher versions BinSets Chimera_Bins_v1 v1X or higher versions Size Standard SST BTO_60 500bp Analysis Method Analysis_HID_310 Analysis_HID_3130 Analysis HID 310 50rfu Analysis HID 3130 50rfu Plot Settings PlotsBT5_4dyes Table Settings Table for 2 Alleles Table for 10 Alleles Panels and BinSets always have to be used whereas the other template files are optional Additional Biotype templates for GeneMapper ID X Software Stutter Chimera_Stutter_v1X or higher version When loading the above mentioned panels the stutter settings will not be accepted Thus the stutter data has to be imported separately Recommended Biotype template files for Genotyper Software are Mentype_Chimera_v1 or higher versions Important Note Import and allele calling with provided template files is only guarantied using GeneMapper ID ID X software If GeneMapper software is applied you may experience import problems using some template files You may have to adjust Panels and Bins with one ore more runs of the allelic ladder on your specific instrument setup Contact us for support support biotype de General procedure for the analysis 1 Check the DNA size standard 2 Check the allelic ladder 3 Check the positive control 4 Check the negative control 5 Analyse and interpret the sample data Mentype Chimera December 2014 LEUGACHNv1en 37
16. Default Dye Set BT5 Run Module Default Protocol Name e g Mentype Chimera Oven Temperature C Default Run Voltage kV Default njection Voltage kV 3 0 Run Time s 1560 PreRun Time s Default njection Time s 10 Data Delay Time s Default Advanced Options Default Deviating from the standard settings the injection time may range between 1 and 20 s depending on the type of sample If reference samples with very high signal intensities are recorded a shorter injection time may be o elected in order to avoid pull up peaks For samples with low DNA content or critical patient samples an njection time of up to 20 s may be necessary Depending on the analysis conditions the run time for Mentype Chimera was modified in order to analyse fragments with lengths of up to 500 bp Click on Save to confirm the settings Mentype Chimera December 2014 LEUGACHNv1en 33 Create Size Standard Go to Library and select Analyze Size Standards and click Create Change the parameters according the table below Parameter Set up Size Standard BTO 550 Dye Color Orange The DNA Size Standard 550 BTO should be used with the following lengths of fragments 60 80 90 100 120 140 160 180 200 220 240 250 260 280 300 320 340 360 380 400 425 450 475 500 525 and 550 bp Click on Save to confirm the settings Create QC Size Calling Protocol Go to Library and selec
17. EUGACHNv1en 25 5 3 Setting up the Data Collection Software Edit the Run Module as follows for the first run In the Module Manager of the Data Collection Software click on New to open the Run Module Editor dialog box Run Module 3kV_10s_500bp Parameter Set up Oven Temperature C Default Poly Fill Volume Default Current Stability LA Default PreRun Voltage kV Default PreRun Time s Default njection Voltage kV 3 0 njection Time s 10 Voltage Number of Steps Default Voltage Step Interval Default Data Delay Time s Default Run Voltage kV Default Run Time s 1560 Deviating from the standard settings the injection time may range between 1 and 20 s depending on the type of sample If reference samples with very high signal intensities are recorded a shorter injection time may be selected in order to avoid pull up peaks For samples with low DNA content or critical patient samples an injection time of up to 20 s may be necessary Depending on the analysis conditions the run time for Mentype Chimera was modified in order to analyse fragments with lengths of up to 500 bp Click on Save As enter the name of the new module e g 3kV_10s_500bp and confirm with OK Click on Close to exit the Run Module Editor Starting the run Place the prepared 96 well plate on the autosampler tray In the Protocol Manager of the Data Collection Software click on New in the Instrument P
18. LEUGACHNv1en 29 Setting up the spectral calibration standards Example for 8 capillaries ABI 3500 Component Volume Hi DiTM Formamide 108 0 ul Matrix standard BT5 9 0 ul Load 12 ul of the mix to a 96 well reaction plate e g position A1 H1 Denaturation for 3 min at 95 C Cool down to 4 C and place samples on the autosampler tray Example for 24 capillaries ABI 3500xL Component Volume Hi Di Formamide 300 0 ul Matrix standard BT5 25 0 yl Load 12 ul of the mix to a 96 well reaction plate e g position A1 H1 A2 H2 and A3 H3 Denaturation for 3 min at 95 C Cool down to 4 C and place samples on the autosampler tray When using a 384 well plate load 10 ul of the mixtures to columns 1 3 and 5 in rows A C E G l K M and 0 Performing a spectral calibration run Place the multi well plate on the autosampler tray Now prepare the instrument and specific spectral calibration run settings Preparation of the instrument Before starting the spectral calibration process ensure that the spatial calibration has been performed This process is necessary if a new capillary array was installed before and is described in detail in the Applied Biosystems 3500 3500xL Genetic Analyzers User Guide Preparation of dye set BT5 Prior to the spectral calibration a dye set for the matrix standard BT5 needs to be setup 1 To create a new dye set go to Library and select Analyze followed by Dye Sets and cl
19. UGACHNv1en 1 Description of Mentype Chimera Table 1 Locus specific information of Mentype Chimera Locus Amelogenin X Amelogenin Y D2S1360 0351744 0452366 0552500 065474 0751517 0851132 01092325 0125391 D18551 D2182055 SE33 ACTBP2 GenBank accession M55418 M55419 608130 608246 608339 608468 608540 618365 608685 608790 608921 118333 627274 NG000840 Repeat motif of the reference allele TGTC o TATC TA TCTA TCA TCTA 2 ATAG o ATTG ATAG gt AGAls TGA 2 CAAA CAAA GAAA gt CTA o TCA TCTAlp TCTGTCTA GAT AGAT AGAC s AGAT AGAA a CTAA CTA CTAT TAT CTATI3 TAT CTAT 4 CAT CTAT AA AAAG 16 Reference Allele allele range 23 19 32 16 13 22 12 9 15 12 9 18 17 11 20 17 14 31 20 12 1 27 12 6 23 19 3 13 28 13 5 3 42 24 16 1 39 25 2 3 50 Table 1 shows STR loci with respective repeat motifs and alleles that are concordant with the guidelines for the use of microsatellite markers of the International Society for Forensic Genetics ISFG Bar et al 1997 The nomenclature for STR loci 0851132 and 0125391 is in accordance with Hering and M ller 2001 for loci 0452366 und 065474 with Becker et al 2007 for locus 01052325 with Wiegand et al 1999 and the nomenclature for locus 0751517 is in accordance with Wiegan
20. aintenance on the Dashboard of the 3500 Series Data Collection Software 2 The number of wells in the spectral calibration plate and their location in the instrument must be specified 3 Select Matrix Standard as a chemistry standard and BT5 for dye set 4 Optional Enable Allow Borrowing 5 Click Start Run Mentype Chimera December 2014 LEUGACHNv1en 31 Cal bewtedi Data m Ta BA C7 u a E Fig 6 Electropherogram of spectral calibration with matrix standard BT5 on an ABI 3500 Matrix check The quality value Q value of each capillary must be greater than 0 8 and the condition number range C value must be between 1 and 20 Check the matrix samples for a flat baseline As shown in the figure above there should be five peaks with peak heights of about 1000 5000 RFU Y axis in each matrix sample optimal range 2000 4000 RFU A successful calibration will be displayed in green in Overall and for each capillary If all capillaries have passed the test Accept Results If calibration failed Reject Results and refer to spectral calibration troubleshooting of Applied Biosystems 3500 3500xL Genetic Analyzer User Guides 6 2 Sample preparation Component Volume Hi Di Formamide 12 0 ul DNA Size Standard 550 BTO 0 5 ul prepare 12 ul of the mix formamide DNA size standard for all samples add 1 ul PCR product diluted if necessary or allelic ladder Denaturation for 3 min at 95 C Co
21. allelic ladder is recommended Scaling Horizontal 70 480 bp Vertical Depending on signal intensity Mentype Chimera December 2014 LEUGACHNv1en Figure 8 e eo N st a M la i5 L N N 2 81 81 e a a lt eo a 8 st 8 lt 2 a Re ki _ EN i N n Za 5 t N X 8 8 na 8 8 c E e 8 to e io e 2 T a de a E Si a e gt lt x 8 8 8 8 8 8 8 8 8 8 8 8 8 8 59558 8 582 8 amp 8 amp Fig 8 Electropherogram of the Mentype Chimera using 500 pg Control DNA XY5 Analysis was performed on an ABI PRISM 3130 Genetic Analyzer with the DNA Size Standard 550 BTO Allele assignment was performed using the GeneMapper ID Software and the Mentype Chimera template file Erz 15 39
22. alyse fragments with lengths of up to 500 bp Mentype Chimera December 2014 LEUGACHNv1en 3 4 Analysis parameter analysis method The recommended analysis parameters are Analysis Range Start 2000 Stop 10000 Data Processing Baseline Checked ulticomponent Checked Smooth Options Light Peak Detection Peak Amplitude Thresholds BRE G R 0 in Peak Half Width 2 pts Polynominal Degree 3 Peak Window Size 11 pts Size Call Range in 60 ax 550 Size Calling Method Local Southern Method Split Peak Correction The peak amplitude threshold cu None off value corresponds to the minimum peak height that will be detected by the GeneScan or GeneMapper ID software Thresholds are usually 50 200 RFU and should be determined individually by the laboratory Recommendation The minimal peak height should be three times as high as the background noise of the baseline Point alleles i e alleles with at least 1 bp difference to the next integer allele may occasionally not be distinguished For improved peak di Mentype Chimera etection minimise the Peak Window Size further December 2014 LEUGACHNv1 en 16 4 Electrophoresis using the ABI PRISM 3100 Avant 3100 Genetic Analyzer For detailed instructions on instrument setup spectral calibration application of the ABI PRISM 3100 Data Collection Software version 1 0 1 or 1 1 and the GeneScan software refer to t
23. d and Klintschar 2002 Allele ranges include all known alleles of the National Institute of Standards and Technology NIST as at 12 2008 and the current literature Table 2 Chromosomal mapping for Mentype Chimera Locus Amelogenin X Amelogenin Y D2S1360 0351744 0452366 0552500 065474 0751517 0851132 01092325 0125391 D18551 02152055 SE33 Chromosomal mapping Xp22 1 22 3 Yp11 2 2p24 p22 3p24 4p16 15 2 5411 2 6021 22 7931 33 8923 1 10p12 12p13 2 18q21 3 21q22 6q14 2 Mentype Chimera December 2014 LEUGACHNv1en Kit content Mentype Chimera PCR Amplification Kit 100 Reactions Nuclease free water Reaction mix A Primer mix Multi Taq2 DNA polymerase Control DNA XY5 2 ng ul DNA Size Standard 550 BTO Allelic ladder Ordering information Mentype Chimera Mentype Chimera Mentype Chimera Mentype Chimera Storage 3 0 ml 500 ul 250 ul 40 yl 10 ul 50 ul 25 ul 25 reactions Cat No 100 reactions Cat No 400 reactions Cat No 1000 reactions Cat No 45 13210 0025 45 13210 0100 45 13210 0400 45 13210 1000 Store all components at 20 C and avoid repeated thawing and freezing Primer mix and allelic ladder must be stored protected from light The DNA samples and post PCR reagents allelic ladder and DNA size standard should be stored separately from PCR reagents The expiry date is indicated on the kit cover Additionally required reagents Additi
24. ed products depends on the device type the conditions of electrophoresis as well as the DNA size standard used Due to the complexity of some STR loci size determination should be based on evenly distributed references The DNA Size Standard 550 BTO shall thus be used with the following lengths of fragments 60 80 90 100 120 140 160 180 200 220 240 250 260 280 300 320 340 360 380 400 425 450 475 500 525 and 550 bp 200 4200 4000 800 600 400 200 60 0 80 0 100 0 120 0 140 0 160 0 180 0 200 0 220 0 240 0 260 0 280 0 300 0 320 0 340 0 360 01380 0400 0 425 0 450 0 475 0 500 0 525 0 550 0 90 0 250 0 Fig 7 Electropherogram of the DNA Size Standard 550 BTO fragments with lengths in bp 300 400 500 Note The provided template files for the DNA size standard SST BTO_60 500bp can be applied for the evaluation and analysis of the Mentype Chimera using the GeneMapper ID or ID X Software Mentype Chimera December 2014 LEUGACHNv1en 36 7 1 Biotype template files Allele allocation should be carried out with suitable analysis software e g GeneMapper ID ID X or Genotyper software in combination with the Mentype Chimera template files from Biotype Biotype template files are available on our homepage www biotype de for download or as CD ROM on request Recommended
25. emarks of Applied Biosystems LLC Under the law of Europe POP 4 is a registered trademark of Applied Biosystems LLC POP 4 is registered as trademark of Life Technologies Corporation in the US The PCR is covered by patents Patentees are Hoffmann La Roche Inc and F Hoffmann La Roche Roche Mentype Chimera December 2014 LEUGACHNv1en 8 Protocols for PCR amplification electrophoresis and analysis 2 PCR amplification 2 1 Master mix preparation The table below shows the volumes of all PCR reagents per 25 ul reaction volume including a sample volume of 1 0 ul template DNA The number of reactions to be set up shall be determined taking into account positive and negative control reactions Add one or two reactions to this number to compensate the pipetting error Component Volume Nuclease free water 16 1 ul Reaction mix A 5 0 ul Primer mix 2 5 ul Multi Taq2 DNA Polymerase hot start 2 5 U ul 0 4 ul Volume of master mix 24 0 pl contains dNTPs BSA All components should be mixed vortex and centrifuged for about 10 s before preparing the master mix The volume of DNA applied to the assay depends on its concentration For reference samples 1 ul is mostly sufficient For critical patient samples the amount of template can be increased appropriately Fill up the final reaction volume to 25 ul with nuclease free water Generally DNA templates shall be stored in nuclease free water or in diluted TE buffer
26. eneration Prior to conducting DNA fragment size analysis with the filter set G5 a matrix with five fluorescent labels 6 FAM BTG BTY BTR and BTO must be generated Color Matrix standard Blue B 6 FAM Green G BTG Yellow Y BIY Red R BTR Orange 0 BTO Five electrophoresis runs shall be conducted one for each fluorescent label 6 FAM BTG BTY BTR and BTO use the same conditions as for samples and allelic ladders of the Biotype test kit to generate suitable matrix files Matrix sample Component Volume atrix sample 1 Hi Di Formamide 12 0 p atrix standard 6 FAM 1 0 atrix sample 2 Hi DiTM Formamide 12 0 standard BTG 1 0 atrix sample 3 Hi DiTM Formamide 12 0 p atrix standard BTY 1 0 trc sampled Hi DiTM Formamide 12 0 p atrix standard BTR 1 0 atrix sample 5 Hi DiTM Formamide 12 0 p atrix standard BTO 1 0 Denaturation for 3 min at 95 C Cool down to 4 C and place samples on the autosampler tray Create a Sample Sheet choose 5 Dyes and enter a sample designation Mentype Chimera December 2014 LEUGACHNv1en Injection list for matrix generation Parameter Set up Module File GS STR POP 4 1 ml G5 Matrix File NONE Size Standard NONE Injection s 5 Injection kV 15 0 Run kV 15 0 Run C 60 Run Time min 24 Prepare matrix standards always without DNA Size Standard BTO Analysis of the matrix samples Run the GeneMapper software File
27. enetic distance Genetics 76 379 390 Szibor R Edelmann J Hering S Plate l Wittig H Roewer L Wiegand P Cali F Romano V Michael M 2003 Cell line DNA typing in forensic genetics the necessity of reliable standards Forensic Sci Int 138 37 43 Wiegand P Lareu M V Sch renkamp M 1999 0185535 0151656 and 01052325 three efficient short tandem repeats for forensic genetics Int J Legal Med 112 360 363 Wiegand P Klintschar M 2002 Population genetic data comparison of the repeat structure and mutation events of two short STRs nt J Legal Med 116 258 261 Mentype Chimera January 2013 48 11 Explanation of Symbols Manufacturer Date of manufacture 2 E rr o Batch code Contains sufficient reagents for lt N gt tests V Gonsult instructions handbook for use Use by Temperature limitations B gt 5 Catalogue number lt In Vitro Diagnostics Mentype Chimera January 2013 Notes Mentype Chimera January 2013 49 50 Notes Mentype Chimera January 2013
28. he ABI PRISM 3100 Avant 3100 Genetic Analyzer User s Manual For systems with Data Collection Software 2 0 or 3 0 refer to chapter 5 The system with 4 capillaries is named ABI 3100 Avant and the system with 16 capillaries is named ABI 3100 The virtual filter set G5 shall be used for combined application of the five fluorescent labels 6 FAM BTG BTY BTR and BTO the matrix standard will be called BT5 hereinafter Material Capillary 36 cm Capillary Array for 3100 Avant 3100 Polymer POP 4 Polymer for 3100 Buffer 10x Genetic Analyzer Buffer with EDTA 4 1 Spectral calibration matrix generation Proper spectral calibration is critical to evaluate multicolour systems with the ABI PRISM 3100 Avant 3100 Genetic Analyzer and shall be done prior to conducting fragment length analysis with the five fluorescent labels 6 FAM BTG BTY BTR and BTO The calibration procedure creates a matrix which is used to correct the overlapping of fluorescence emission spectra of the dyes Spectral calibration comprises the following steps Preparation of the spectral calibration standards Loading the standards to the 96 well reaction plate one sample per capillary Entering the plate composition Performing a spectral calibration run and checking the matrix Setting up the spectral calibration standard Example for 4 capillaries ABI 3100 Avant Component Volume Hi Di Formamide 60 0 ul Matrix standard BT5 5 0 ul Load 12
29. ick Create 2 Enter a Dye Set Name e g BT5 3 Select Matrix Standard as a chemistry and AnyDye Template as a Dye Set Template 4 Disable Purple in the field Arrange Dyes Ensure that all other colors are enabled 5 Under Calibration Peak Order the colors need to be arranged as Follows 5 blue 4 green yellow 2 red and 1 orange 6 Do not alter the Parameter settings T Click Save to confirm the changes Mentype Chimera December 2014 LEUGACHNv1en 30 Setup a Dye Set lt Dye Set Name BTS Chemistry Matrix Standard Dye Set Template AnyDye Template v Arrange Dyes Sn o EE NN Reduced Selection v wl vi v Calibration Peal Order 5 10 al 21 7 Parameters The parameters will be used for instruments configured with 36cm capillary array and polymer POP4 Matrix Condition Number Upper Lim t 20 0 Locate Start Point After Scan 300 Before Scan 5000 Limit Scans To Sensitivity Minimum Quality Score Notes Matrix Std BTS multi cap Fig 5 Setup for dye set BT5 In the Protocol Manager of the Data Collection Software click on New in Instrument Protocol to open the Protocol Editor dialog box Performing a spectral calibration run Once the multi well plates containing the spectral calibration mixture is placed in the autosampler tray the spectral calibration process can be started 1 To access the Spectral Calibration screen select M
30. in order to avoid pull up peaks For samples with low DNA content or critical patient samples an injection time of up to 20 s may be necessary Depending on the analysis conditions the run time for Mentype Chimera was modified in order to analyse fragments with lengths of up to 500 bp Click on Save As enter the name of the new module e g 3kV 10s 500bp and confirm with OK Click on Close to exit the Run Module Editor Starting the run Place the prepared 96 well plate on the autosampler tray Run the ABI PRISM 3100 Data Collection Software n Plate View click on New to open the Plate Editor dialog box Enter a name of the plate Select GeneScan Select 96 Well as plate type and click on Finish Mentype Chimera December 2014 LEUGACHNv1en 20 Plate Editor Parameter Set up Sample Name Enter name for the samples Dyes 0 Colour Info Ladder or sample Project Name e g 3100_Project1 Dye Set G5 Run Module 3kV_10s_500bp Analysis Module 1 DefaultAnalysis gsp parameter see above Complete the table in the Plate Editor and click on OK Click into the column header to select the entire column and select Edit Fill Down to apply the information of the selected samples Link your reaction plate on the autosampler tray with the created plate ID and start the run On completion of the run view data as Color Data in Array View of the 3100 Data Collection Software or as Analyzed Sample Files unde
31. ion of the Multi Taq2 DNA Polymerase 94 C 30s 60 C 120s 30 cycles 72 C 758 68 C 60 min 10 o0 hold Optional Recommended for small amounts of DNA Temperature Time 94 C 4 min hot start for activation of the Multi Taq2 DNA Polymerase 94 C 30s 60 C 120s 32 cycles 72 758 68 C 60 min 10 C co hold Note If thermal cyclers with rapid heating and cooling steps gt 2 C s are used ramping should be adjusted to 2 C s in order to provide an optimal kit balance Very small amounts of DNA may result in statistical dropouts and imbalances of the peaks Increasing numbers of PCR cycles raise the risk of cross contamination caused by minimal amounts of impurities Furthermore unspecific amplification products could appear Mentype Chimera December 2014 LEUGACHNv1en 10 3 Electrophoresis using the ABI PRISM 310 Genetic Analyzer For general instructions on instrument setup matrix generation and application of the GeneScan or GeneMapper ID software refer to the ABI PRISM 310 Genetic Analyzer User s Manual Electrophoresis using the GeneScan software is described below The virtual filter set G5 shall be used for combined application of the five fluorescent labels 6 FAM BTG BTY BTR and BTO the matrix standard will be called BT5 hereinafter Material Capillary 47 cm 50 um green Polymer POP 4 for 310 Genetic Analyzer Buffer 10x Genetic Analyzer Buffer with EDTA 3 1 Matrix g
32. les N 4 peaks are caused by a loss of a repeat unit during amplification of tetranucleotide STR motives caused by slippage effects of the Taq DNA Polymerase Interpretation of those peaks should be done in accordance with the template files of the Genotyper and GeneMapper ID ID X software Template independent addition of nucleotides Because of its terminal transferase activity the Multi Taq DNA Polymerase tends to add an adenosine radical at the 3 end of the amplified DNA fragments The artefact peak is one base shorter than expected 1 bp peaks All Biotype primers are designed to minimise these artefacts Artefact formation is further reduced by the final extension step of the PCR protocol at 68 C for 60 min Peak height of the artefact correlates with the amount of DNA Laboratories should define their individual limits for analysis of the peaks Artefacts Room temperature may influence the performance of PCR products on multi capillary instruments shoulder peaks or split peaks occur Furthermore automated assignment could be influenced in some cases If these effects occur we recommend injecting the sample again at higher room temperature and maybe using more than one allelic ladder sample per run Influence of polymers The Mentype Chimera kit was validated and certified for the analysis on POP 4 polymer The use of other polymers e g POP 7 or POP 6 might influence the run behaviour of specific PCR products Further
33. me Enter name for the matrix samples Priority e g 100 Instrument Protocol 1 Spectral36_POP4_BT5 setting described before Click into the column header to select the entire column select Edit Fill Down to apply the information to all selected samples and click on OK n the Run Scheduler click on Find All select Link to link the reaction plate on the autosampler to the newly created plate record position A or B and start the run EI ut cut ETT LI Fig 4 Electropherogram of spectral calibration with matrix standard BT5 on an ABI 3130 Matrix check The quality value Q value of each capillary must be greater than 0 95 and the condition number range C value must be between 1 and 20 Check the matrix samples for a flat baseline As shown in the figure above there should be five peaks with peak heights of about 1000 5000 RFU Y axis in each matrix sample optimal range 2000 4000 RFU If all capillaries have passed the test the last calibration file for the Dye Set AnybDye is activated automatically in the Spectral Viewer Rename the calibration file e g BT5 Date of calibration using the respective button If calibration was not successful try to re inject the samples with higher injection voltage or injection time Editing of the Spectral Run Module will be necessary You Mentype Chimera December 2014 LEUGACHNv1en 24 can re inject the same samples up to three times Otherwise use
34. min at 95 C Cool down to 4 C and place samples on the autosampler tray Example for 16 capillaries ABI 3130xl Component Volume Hi DiTM Formamide 204 0 ul Matrix standard BT5 17 0 yl Load 12 ul of the mix to a 96 well reaction plate e g position A1 H1 and A2 H2 Denaturation for 3 min at 95 C Cool down to 4 C and place samples on the autosampler tray Performing a spectral calibration run Place the 96 well plate on the autosampler tray In the Protocol Manager of the Data Collection Software click on New in Instrument Protocol to open the Protocol Editor dialog box Instrument Protocol for spectral calibration Protocol Editor Setup Name User enter name Type SPECTRAL Dye Set Any5Dye Polymer User POP 4 Array Length User 36cm Chemistry Matrix Standard Run Module Default enter a name for the run module Depends on the type of polymer and length of capillary used Click on OK to leave the Protocol Editor dialog box In the Plate Manager of the Data Collection Software click on New to open the New Plate Dialog box Plate Editor for spectral calibration I New Plate Dialog Set up Name e g Spectral_BT5_date Application Spectral Calibration Plate Type 96 Well Owner Name Operator Name Click on OK A new table in the Plate Editor will open automatically Mentype Chimera December 2014 LEUGACHNv1en 23 Plate Editor for spectral calibration II Parameter Set up Sample Na
35. more background noise might increase through different behaviour of free fluorescent dyes Mentype Chimera January 2013 44 9 Population genetic data Most important population genetic data of the STR markers are listed in table 7 10 The formula to calculate Polymorphism Information Content PIC was published by Botstein et al 1980 Expected Heterocygosity HET by Nei and Roychoudhury et al 1974 and Power of Discrimination PD refers to Jones et al 1972 All formulas are suitable for autosomale markers 1 2 25 rin i 1 i j i 1 2 PD 1 2 12 Tabelle 7 Population genetic data Marker D2S1360 Marker D3S1744 Marker D4S2366 Allele Allele frequency Allele Allele frequency Allele Allele frequency 19 0 007 13 0 007 9 0 347 20 0 126 14 0 104 10 0 179 21 0 060 15 0 053 11 0 074 22 0 309 16 0 100 12 0 147 23 0 142 17 0 319 13 0 168 24 0 098 18 0 197 14 0 074 25 0 086 19 0 130 15 0 011 26 0 093 20 0 067 27 0 035 21 0 023 28 0 023 29 0 012 PIC 0 790 PIC 0 760 30 0 002 PD 0 943 PD 0 919 31 0 005 HET 0 792 HET 0 795 32 0 002 PIC 0 820 PD 0 955 HET 0 856 Mentype Chimera January 2013 Tabelle 8 Population genetic data Marker D5S2500 Marker D6S474 Marker 0751517 Allele Allele frequency Allele Allele frequency Allele Allele frequency 9 0 007 8 0 246 16 0 007 0 0 084 4 0 212 17 0 007 1 0 313 5 0 154 18 0 049 2 0 161 6 0 285 19 0 120 3 0
36. ol down to 4 C and place samples on the autosampler tray Since injections take place simultaneously on all capillaries 8 or 24 samples must be pipetted on the plate of multi capillary analyzers If fewer samples are analysed empty positions need to be filled with 12 ul Hi DiTM Formamide To ensure a reliable allelic assignment on multi capillary analyzers several ladders should be run Room temperature may influence the performance of PCR products on multi capillary instruments so that shoulder peaks or split peaks occur especially at low temperatures Pay attention to keeping ambient conditions as recommended by the instrument manufacturer Optimal will be a stable room temperature gt 22 C Mentype Chimera December 2014 LEUGACHNv1en 32 Signal intensities Options to increase the signal intensity Reduce the volume of the DNA Size Standard 550 BTO to peak heights of about 500 relative fluorescent units RFU Purify the PCR products before starting the analysis 6 3 Setting up a run For the first run using the Mentype Chimera Kit you will need to setup a number of protocols within the 3500 Series Data Collection Software Create Instrument protocol Go to Library and select Analyze Instrument protocol and click Create Change the parameters according the table below Instrument protocol for Mentype Chimera Parameter Set up Application Type HID Microsatellite Capillary Length Default Polymer
37. onal reagents required in order to use the Biotype PCR Amplification Kit Reagent Hi DiTM Formamide 25 ml Matrix Standards BT5 single capillary instruments 5x25 ul Matrix Standards BT5 multi capillary instruments 25 yl Matrix Standards BT5 multi capillary instruments 50 yl Mentype Chimera Supplier Life Technologies Corporation Biotype Diagnostic GmbH Biotype Diagnostic GmbH Biotype Diagnostic GmbH December 2014 Order number 4311320 00 10411 0025 00 10421 0025 00 10421 0050 LEUGACHNv1en Warnings and safety instructions The PCR Amplification Kit contains the following potentially hazardous chemicals Kit component Chemical Hazards Reaction mix Sodium azide NaN toxic if swallowed develops toxic gases when it gets in contact with acids Observe the Material Safety Data Sheets MSDS for all Biotype products which are available on request Please contact the respective manufacturers for copies of the MSDS for any additionally needed reagents Quality assurance All kit components undergo an intensive quality assurance process at Biotype Diagnostic GmbH Quality of the test kits is permanently monitored to ensure unrestricted usability Please contact us if you have any questions regarding quality assurance Trademarks and Patents Mentype and Chimera are registered trademarks of Biotype Diagnostic GmbH ABI PRISM GeneMapper GeneAmp and Applied Biosystems are registered trad
38. or R Data Start At 1000 No File Selected for O Data Start 1000 Points 100000 Create p Matrix Result B R o 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 10 0000 10 0000 Rm Fig 2 Matrix sample selection Enter a Start At value e g 2900 Enter the calculated difference under Points e g 2500 Mentype Chimera December 2014 LEUGACHNv1en w Matrix Editor Matrix Description Matrix Matrix BTS Security Group GeneMapper ID X Security Group Description Matrix Settings Select the Matrix Standard Sample File Number of Dyes 5 5 M Stab_MST_310_FAM_10 05 1 fsa Start At 2900 Stab MST 310 BTG 10 05 1 fsa Start At 2900 Stab MST 310 BTY 10 05 1 fsa Start At 2900 Stab MST 310 BTR 10 05 1 fsa Start At 2900 Stab MST 310 BTO 10 05 1 fsa Start At 2900 Points 2500 Fig 3 New matrix BT5 Calculate the matrix with Create Click on OK to save the new matrix Matrix check Check the new matrix with current samples File gt Add Samples to Project open folder of the respective run Select sample s in the Sample File column select the new matrix in the Sample Table Re analyse your samples There should be no pull up peaks between the dye panels B G Y R 0 with
39. or split peaks occur especially at low temperatures Pay attention to Keeping ambient conditions as recommended by the instrument manufacturer Optimal will be a stable room temperature gt 22 C Signal intensities Options to increase the signal intensity Reduce the volume of the DNA Size Standard 550 BTO to peak heights of about 500 relative fluorescent units RFU Purify the PCR products before starting the analysis Mentype Chimera December 2014 LEUGACHNv1en 4 3 Setting up the Data Collection Software Edit the default run module in Dye Set G5 once for the first run Select Module Editor to open the dialog box Select the appropriate Run Module as template from the GeneScan table Modify the Injection Voltage to 3 kV and the Injection Time to 10 s Run Module 3kV 10s 500bp Parameter Set up Run Temperature C Default Cap Fill Volume Default aximum Current A Default Current Tolerance A Default Run Current A Default Voltage Tolerance kV Default Pre Run Voltage kV Default Pre Run Time s Default njection Voltage kV 3 0 njection Time s 10 Run Voltage kV Default Number of Steps Default Voltage Step Interval Default Data Delay Time s Default Run Time min 26 Deviating from the standard settings the injection time may range between 1 and 20 s depending on the type of sample If reference samples with very high signal intensities are recorded a shorter injection time may be selected
40. oup to all named wells in the plate Click Link the plate for Run and enter Run Name Click Start Run 6 4 Analysis parameter analysis method The recommended analysis parameters are Peak Detection Algorithm Advanced Ranges Analysis Partial Range Start Pt 2000 Stop Pt 10000 Sizing All Sizes Smoothing and Baselining Smoothing Light Baseline Window 51 pts Size Calling Method Local Southern Method Peak Detection Peak Amplitude Thresholds BE ys G R 0 in Peak Half Width 2 pts Polynominal Degree 3 Peak Window Size 11 pts Slope Thresholds 0 0 The peak amplitude threshold cut off value corresponds to the minimum peak height that will be detected by the GeneMapper ID ID X software The thresholds are usually 50 200 RFU and should be determined individually by the laboratory Recommendation The minimal peak height should be three times as high as the background noise of the baseline Point alleles i e alleles with at least 1 bp difference to the next integer allele may occasionally not be distinguished For improved peak detection minimise the Peak Window Size further Mentype Chimera December 2014 LEUGACHNv1en 35 7 Analysis For general instructions on automatic sample analysis refer to the GeneScan or GeneMapper ID or GeneMapper ID X Software User s Manual Note Within the Mentype Chimera the red panel should be faded out Finding the exact lengths of amplifi
41. r D AppliedBio 31 00 DataExtractor ExtractRuns 4 4 Analysis parameter analysis method The recommended analysis parameters are Analysis Range Start 2000 Stop 10000 Data Processing Baseline Checked ulticomponent Checked Smooth Options Light Peak Detection Peak Amplitude Thresholds B YE GT RE 0 in Peak Half Width 2 pts Polynominal Degree 3 Peak Window Size 11 pts Size Call Range Min 60 550 Size Calling Method Local Southern Method Split Peak Correction lone The peak amplitude threshold cut off value corresponds to the minimum peak height that will be detected by the GeneScan or GeneMapper ID software Thresholds are usually 50 200 RFU and should be determined individually by the laboratory Recommendation The minimal peak height should be three times as high as the background noise of the baseline Point alleles i e alleles with at least 1 bp difference to the next integer allele may occasionally not be distinguished For improved peak detection minimise the Peak Window Size further Mentype Chimera December 2014 LEUGACHNv1en 21 5 Electrophoresis using the ABI PRISM 3130 3130xl Genetic Analyzer For detailed instructions on instrument setup spectral calibration or application of the ABI PRISM Data Collection Software version 3 0 and the GeneMapper ID ID X Software refer to the ABI PRISM 3130 3130x Genetic Analyzers Getting Started Guide The system with
42. rotocol window to open the Protocol Editor dialog box Instrument Protocol Protocol Editor Set up Name enter a name Type REGULAR Run Module 3kV 10s 500bp Dye Set Any5Dye parameter see above Click on OK to exit the Protocol Editor Mentype Chimera December 2014 LEUGACHNv1en 26 Prior to each run it is necessary to create a plate definition as follows In the Plate Manager of the Data Collection Software click on New to open the New Plate Dialog box Plate Editor I New Plate Dialog Set up Name e g Plate_BT5_Date Application Select GeneMapper Application Plate Type 96 Well Owner Name Operator Name te Click on OK A new table in the Plate Editor will open automatically Plate Editor II Parameter Set up Sample Name Enter a name for the samples Priori e g 100 Default Sample Type Sample or allelic ladder Size Standard e g SST BTO_60 500bp Pane e g Chimera Panels vi Analysis Method e g Analysis HID 3130 Snp Set User defined 1 3 Results Group 1 select results group Instrument Protocol 1 Run36_POP4_BT5_ 26min setting described before Click into the column header to select the entire column select Edit Fill Down to apply the information to all selected samples and click on OK n the Run Scheduler click on Find All select Link to link the reaction plate on the autosampler to the newly created plate record position A or B and start the run During the run view
43. short tandem repeats STRs with a very high rate of heterozygosity and a balanced allelic distribution Together this significantly increases the chance to identify informative loci for donor recipient discrimination and provides reliability and robustness of chimerism analyses One PCR reaction simultaneously amplifies the autosomal loci 0251360 D3S1744 0452366 D5S2500 065474 0751517 0851132 01052325 0125391 D18S51 02152055 SE33 ACTBP2 and the gender specific locus Amelogenin One primer for each locus is fluorescence labelled with 6 FAM BTG or BTY The detection limit of the Mentype Chimera PCR amplification kit is 200 pg genomic DNA The optimal range under standard conditions is 0 2 1 0 ng DNA The test kit is validated using the GeneAmp PCR System 9700 Aluminium Eppendorf Mastercycler ep S Biometra T1 ABI PRISM 310 Genetic Analyzer and ABI PRISM 3130 Genetic Analyzer applying the 4 polymer Development manufacture and distribution of Biotype products are certified according to DIN EN ISO 9001 2008 Mentype Chimera December 2014 LEUGACHNv1en Content 1 2 LAA VIN ders rcs end 8 9 10 References Electrophoresis using the ABI PRISM 310 Genetic Analyzer Description of Mentype Chimera PCR amplification 2 1 Master mix preparation 2 2 PCR amplification parameter
44. t Analyze QC Size Calling and click Create Change the parameters according the table below Parameter Set up Protocol Name enter a name Size Standard BTO 550 from above Sizecaller Size Caller v 1 1 0 Go to Analysis Settings Peak Amplitude Treshold and disable purple All other colours should be enabled Keep all other settings as Default Click on Save to confirm the settings Create an Assay Go to Library and select Manage Assays and click Create Change the parameters according the table below Parameter Set up Assay Name e g Mentype Chimera Color Default Application Type HID Instrument Protocol e g Mentype Chimera QC Protocols e g BTO 550 Genemapper Protocol could be defined Click on Save to confirm the settings Mentype Chimera December 2014 LEUGACHNv1en 34 Starting the run Place the prepared multi well plate on the autosampler tray In the Dashboard of the Data Collection Software click Create New Plate Go to Define Plate Properties and select Plate Details Change the parameters according the table below Plate Details Property Setup Name e g Mentype Chimera Number of Wells 96 or 384 Plate Type HID Capillary Length 36cm Polymer POP4 Click Assign Plate Contents to confirm the settings Define well position of each sample or ladder for data collection and processing by entering sample names Assign an Assay required a File Name Conventions and a Result Gr
45. the calibration the last calibration file for Dye Set G5 must be activated manually under Tools Set Active Spectral Calibration Rename the calibration file under Set Matrix Name e g BT5 Date of calibration If calibration was not successful try to re inject the samples with higher injection voltage or injection time The editing of the Spectral Run Module will be necessary You can re inject the same samples up to three times Otherwise use more matrix standard for spectral calibration Check the new matrix with your current samples There should be no pull up peaks between the dye panels B G Y O with the new matrix 4 2 Sample preparation Component Volume Hi DiTM Formamide 12 0 ul DNA Size Standard 550 BTO 0 5 yl Prepare 12 ul of the mix formamide DNA size standard for all samples Add 1 ul PCR product diluted if necessary or allelic ladder Denaturation for 3 min at 95 C Cool down to 4 C and place on the autosampler tray co ince injections take place simultaneously on all capillaries 4 or 16 samples must be ipetted on the plate of multi capillary analyzers If fewer samples are analysed the mpty positions must be filled with 12 ul Hi DiTM Formamide To ensure reliable allelic assignment on multi capillary analyzers several allelic ladders should be run Room temperature may influence the performance of PCR products on multi capillary instruments so that shoulder peaks
46. the new matrix Mentype Chimera December 2014 LEUGACHNv1en 14 3 2 Sample preparation Component Volume Hi Di Formamide 12 0 ul DNA Size Standard 550 BTO 0 5 jil prepare 12 ul of the mix formamide DNA size standard for all samples add 1 ul PCR product diluted if necessary or allelic ladder Denaturation for 3 min at 95 C Cool down to 4 C and place samples on the autosampler tray Signal intensities Options to increase the signal intensity Reduce the volume of the DNA Size Standard 550 BTO to peak heights of about 500 relative fluorescent units RFU Purify the PCR products before starting the analysis 3 3 Setting up the Data Collection Software Create a Sample Sheet and enter sample designation Injection list Parameter Set up Module File GS STR POP 4 1 ml G5 Matrix File e g Matrix BT5 Size Standard e g SST BTO 60 500bp Injection s 5 Injection kV 15 0 Run kV 15 0 Run C 60 Run Time min 28 Deviating from the standard settings the injection time may range between 1 and 20 s depending on the type of sample If reference samples with very high signal intensities are recorded a shorter injection time may be selected in order to avoid pull up peaks For samples with low DNA content or critical patient samples an injection time of up to 20 s may be necessary Depending on the analysis conditions the run time for Mentype Chimera was modified in order to an
47. tral Calibration Select 96 Well as plate type and click on Finish Plate editor for spectral calibration II Parameter Set up Sample Name Enter name for the matrix samples Dye Set 65 Spectral Run Module Default enter the name for spectral run module Spectral Parameters MtxStd GeneScan_SetG5_BT5 par parameters created before Click into the column header to select the entire column select Edit Fill Down to apply the information of the selected samples and confirm with OK Link your reaction plate on the autosampler tray with the created plate ID and start the run On completion of the run check in the Spectral Calibration Result dialog box if all capillaries have successfully passed calibration label A If individual capillaries are labelled X refer to AB PRISM 9 Genetic Analyzer User s Manual Click on OK to confirm completion of the run Mentype Chimera December 2014 LEUGACHNv1en 18 Matrix check Select Tools Display Spectral Calibration Dye Set G5 to review the spectral calibration profile for each capillary The quality value Q value must be greater than 0 95 and the condition number C value must be between 1 and 20 Both values must be within the previously determined range Check the matrix samples for a flat baseline There should be five peaks with peak heights of about 1000 5000 RFU Y axis in each matrix sample optimal range 2000 4000 RFU If all capillaries have passed
48. ul of the mix to a 96 well reaction plate e g position A1 D1 Denaturation for 3 min at 95 C Cool down to 4 C and place samples on the autosampler tray Example for 16 capillaries ABI 3100 Component Volume Hi DiTM Formamide 204 0 ul Matrix standard BT5 17 0 ul Load 12 ul of the mix to a 96 well reaction plate e g position A1 H1 and A2 H2 Denaturation for 3 min at 95 C Cool down to 4 C and place samples on the autosampler tray Mentype Chimera December 2014 LEUGACHNv1en Performing a spectral calibration run First of all the parameter file for DyeSetG5 must be modified once to achieve Successful calibration with the Data Collection Software version 1 0 1 or 1 1 Spectral parameter To change settings in the parameter file go to the following path D AppliedBio Support Files Data Collection Support Files CalibrationData Spectral Calibration ParamFiles Select MtxStd Genescan_SetG5 to open the PAR file Change Condition Bounds Range to 1 0 20 0 Select File Save As to save the parameter file under a new name e g MtxStd Genescan_SetG5_BT5 par Always use this parameter file for spectral calibration runs using Biotype matrix standard BT5 Plate Editor for spectral calibration I Place the 96 well plate on the autosampler tray Run the ABI PRISM 3100 Data Collection Software n Plate View click New to open the Plate Editor dialog box Enter a name of the plate Select Spec
49. vz B v sy ey EH 821192 tz 22 02 8 x N 0 E a Sa e T wa 8S Se o Bs oz m b y 00 002 00 La 2574 PLIS 09615 x tsziq wsad 11518 m January 2013 Mentype Chimera the Mentype Chimera template file 40 Table 4 Fragment lengths of he Mentype Chimera allelic ladder analysed on an ABI PRISM 3130 Genetic Analyzer with 4 polymer blue panel Marker allele Size bp Marker allele Size p bod Marker allele Size bp JA Amelogenin 6 FAM 0125391 6 FAM 065474 6 FAM X 77 15 213 3 354 11 12 Y 80 16 217 16 3 4 358 17 221 17 3 5 362 0751517 6 FAM 18 226 18 3 6 366 16 08 14 15 19 230 191 193 7 370 17 12 20 234 20 3 8 374 18 16 21 238 9 378 19 20 22 242 20 24 23 246 0452366 6 FAM 21 28 24 250 9 429 9 2 22 32 25 254 0 433 10 2 23 36 26 258 27 1 437 24 40 11 2 440 25 44 0251360 6 2 441 26 48 19 281 3 445 27 52 20 285 4 449 28 55 29 1 289 5 454 22 293 0351744 6 FAM 23 297 13 65 24 302 14 69 25 306 15 73 26 310 16 77 27 314 17 82 28 318 18 86 29 322 19 90 30 326 20 94 31 330 21 98 22 32 334 Mentype Chimera January 2013 Table 5 Fragment lengths of the Mentype Chimera allelic ladder analysed on an ABI PRISM 3130 Genetic Analyzer with POP 49 polymer green panel Marker allele Size bp dad Marker allele Size bp Marker
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