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Interpret software - Oxford Gene Technology

1. Apply Changes and Close Automatically normalise during protocol Some feature extraction software will normalise the data If the data has been normalised by the feature extraction software then the tick box should be ticked off If the data has not been normalised then the tick box should be ticked and the CytoSure software will normalise the data 990055 Version 11 112 Normalisation Method Assuming that the CytoSure software Is carrying out the Normalisation then for most arrays the Global Normalisation should be selected However with some custom arrays where there is the possibility that an aberration could cover are large proportion of the probes on the array then the local normalisation should be selected Global Normalisation Setting This setting should not need to be changed Local normalisation method If local normalisation is selected then the region of the genome that is used for normalisation needs to be selected This should be a region that is expected to have a log2 ratio 0 The chromosome should be selected and then the region can be selected by band or genomic position Then click on Add region Probe filtration setting Probe filtration is the ability to remove certain probes from the analysis because they may have low signal or certain other non desirable characteristics 990055 Version 11 113 Options Classification Report Generation
2. ee ea ee e IX I TE neces cone soar z1 2 Array Design Syndrome Plus v2 a Hau Osea Sample Details yn 1 Chr Start Stop Mos B m 1 45075972 45083809 0 0 17 vn 1 109157762 109565904 0 5 14 di ALLE delet 59 210 aj 7256235 7790006 0 53 7 E 9 Ren 784566 0 27 8 X x 8 48598672 43599699 O 0 6 L Duplication Deletion zc Syndrome Region 9988076 20012010 mum CNW Region 2119073 WO 8 PR Detection Method 5 90 506 Tum IUE P Value 53214339 53604264 Comments LIE NE NI M lM M This is an editable region where comments can be type in 1 1 N E hole Genome ene Ee 9 i Figure 55 Dialogue box to add comments on a particular aberration Editing and annotating the aberration list in Genomic view In Genomic view navigate to an aberration and right click see figure 55 to reveal the following options Edit Annotate the aberration as described in 5 4 2 Delete Delete the aberration from the aberration list Ensembl View the aberration in Ensembl UCSC View the aberration in UCSC 990055 Version 11 65 ogt Genomic view Table View Aberrations Chromosome 15 Chromosome Section 17 70107 7MB 18 993737Mb 20 2856398 Mb 21 579059Mb Delete view Region in Ensembl EU E pi T LUC SC 660 probes displayed Figure 56 Editing an aberration in Genomic view 5 5 8 C
3. 87 Use of the software in Workflow mode euIWiLi prc e 87 Conlacr detal S repere EE ee eee ae EENET 88 W TeriBiufeduri eec E E E 88 Version 11 3 Introduction The CytoSure Interpret software is a specialist Cytogenetic software package designed for the analysis of array CGH by Cytogenetists It is designed typically to support experiments where a reference experimental design has been carried out A reference experimental design is defined as follows Array 1 Sample 1 versus Reference Array 2 Sample 2 versus Reference CytoSure analysis software also supports a loop experimental design For further details see section 6 An overview of a typical analysis is shown in the flowchart below 990055 Version 11 4 Oxford Gene Technology xt file Section described Import txt file using File gt Import Initial view of data Run CBS to identify aberrations 5 1 5 2 Manually verify aberrations 22 Classify the aberrations identified 9 4 4 9 4 3 File gt Save cgh file 9 4 4 Save aberrations and annotation to database database Export results for printing e He The software can be operated in two distinct modes Standard software mode In this mode all the software options and settings can be altered as the user works through the analysis 990055 Version 11 C1 Workflow mode Workflow mode simpl
4. H U S se ge ar T re A na a OE id ia an i al a ae Iis E era pue Pe pra tal Be Ane 3 1 PO NE ARTE ae ae ane Olt E i ED Sample Information Miss age Pp aA RR PRO e DEREN GE SRR EDE DU Ae Rn Sample ID y ON f Experiment Sample M vs Reference M en 9 H 5 N Sample Details z Chromosome Section S d 163 524658Mb 163 752841Mb 163 981025Mb 164 209209Mb pi ee Phenotype Details EI Inv K v E v Ex N n i vn JN Aberrations Ve i Chr Start Stop Mbi s j e E ly O Inv x Lose JL_Remove J Bet J 990055 Version 11 Figure 46 Locate the aberration 2 Zoom in All probes shown on the screen should be within the aberration There should be no flanking probes visible otherwise these will be included in the export Genome Genomic View Table View Aberrations Database Management Population Analy Phenotype Details Ld N n N T N N Z e z e e D Irw x m 717003 m3 TT SS Ss es ET m va m LP w n w m ass Gans om ome n meee mmc eo B N o N gt 25 d lt Z Y rooot6zzszsz X 10 probes displayed Figure 47 Expand so only the data points in the aberration are visible on the screen 3 Click Add Repeat for other aberrations in the chromosome It is important to include all aberrations on the same chromosome see discussion below 990055 Version 11 56 Genome Genomic Vie
5. egrqwer earawer enome lt 02 Dec 200 12 Dec 200 147306825 147644891 0 338 Unclassified 79 16 Feb 200 147306825 147644891 0 338 Unclassified 100 100 Variation 67 U FebzUi 2 mae 4296 02 Dec 200 4 18497723 18658198 2 Jan 200 4 18497723 18658198 16 Feb 200 4 18497723 18658198 wgeceqwane 0385760 70622775 0 237 CNV Geen 100 X Wans O oo Raman Os Feb 200 6 2556676 32660134 17 Feb 200 17 Feb 2008 768i 17878 02 Dec 200 8 7256235 79006 02 Jen 200 8 7256235 79006 d20ec20 B 7256235 7790006 16 Feb 200 8 7256235 790006 C Program Files CytoSure Table View O Ideogram View Group data by Submission Aberration Refresh Commit Chr Start Stop Siee Mb Classification CNV Gain Loss CNW Regions Syndrome Important Decipher Aberrations P Value ScanDate Arra 1 45075972 45083809 0 0070 Unclassified 0 PTCH2 4 77173432 08 14 2008 0 01117044 08 14 2008 0 01113993 08 14 2008 Patient Pati l 0 00962261 08 14 2008 Patient Pati Variation 42 U Patient Pati 1 95011343 195065625 1 05 2008 1 ae7si472 46328513 177 Undassfied EN 2 eei20i08e 241202495 0 0010 ndassfed 100 0 EqS7monos CAPNIG Patient Pai Undessfied 0 1 o 7 779058 08 14 2008 Unclassified
6. a k lt ME E a i i i i E 2 1 ii i 0 Wetten ayes RE mor y RU PCL NEEL EBB LOONIE AER A TEE rag Sample ID N i S Experiment Sample M vs Reference M 2 ce a N i E lt Sample Details 9 i 7 8 Chromosome Section E i 39 007163Mb 39 410209Mb 39 813255Mb 30 216302Mb 2 1 j Phenotype Details L1 NE 0348 42804C8 1 363 S RC 60 0100 Inv TES ESSI eee E v i 1 8 39451608 39451667 mr pn 211 8321 vig lt e fi 0 x i ciat dus eL Malt arte Tad a kar E Gi ie 44 8851 N i 15 216 e Aberrations L i 8 E a 1 0 20918377114834275 Chr Start Stop Mb E j18 E a 0 6706150180322552 hy 2 o i 19 1 20 518 37 211 8321 244 8851 134 probes displayed TA NP ii ED C OO Gal B S ls EL lH 1 ln 1 lT 1E Eiio MILL II ij E vL S al T t 8 EES i E AL ENTE Bl l 2 Whole 7 gt maT Genome lt nmm Em Figure 7 Hovering over a probe The following information is displayed e name of the probe location of the probe within its associated chromosome mean red and green signal values log ratio of these values any other calculated ratios if available individual signal values for each spot on the array contributing to the mean signal values X The user is able to navigate the chromosome genome in the same manner as in the chromosome overview B with the addition of being able to traverse the signal ratio axis as well as the l
7. 15 11 03 2008 1 5 11 03 2008 1 3 Thu Mar 26 10 S softwarelC Syndrome Plus CJ 1A A C8 scan 11 03 2008 1 0 Thu Mar 26 10 Siisoftware c Syndrome Plus VA 1A A0 C8 scan 08 14 2008 1 20 Thu Mar 26 11 P test XDBiL Syndrome Plus 00 1A A0 C8 scan 09 14 2008 1 20 Thu Mar 26 11 P itest XDBIL Syndrome Plus MAR TESST asdf asdof asd igasdgsgsr 00 LA A0 CB scan 08 14 2008 1 20 Thu Mer 26 11 Pritest XDBIL Syndrome Phas MAR TESST lasd asdofasd gasdgsqsr 00 1A A0 CB scan 09 14 2008 1 20 Fri Mar 27 111 Prest XDEVL Syndrome Plus 1A A0 C8 scan 12 15 2008 1 27 Fri Mar 27 111 PAD184 cghld Syndrome Plus 1A AQ CB scan 08 14 2008 1 0 Mon Mar 30 1 PtestiXDE Syndrome Plus 1A A0 C8 scan Y das re 16 53214336 3604264 0 389 dessified 8 0 Patient Pati 0 00627653 Loss 15 s95970 presori p 464 dassified 12 4 Wariation_30 Congenital d 85329453 Loss 15 1171815 1190973 9 019 dashed 100 100 Wariation_30 Prader Wilk Potiert Pati 0 00143638 Ga 9 jass9e672 43599699 0 0010 dassified 100 100 variation_46 Patient 0 0 055 05 05 15 19388076 0012010 0 023 dassihed 100 1009 Vanekion 87 Potiert Pati 0 00558106 Gs 9 509607 784666 0 275 dassified 100 100 variation_34 Patient 62741425 Loss 1 45075972 5083809 0 0070 dessified 0 W prow 371
8. Aberrations Chr Start Z Y FOOOIL6ZZSZSZ L S g S lez 2 RP11 44K6 Toton O lt 0mMo On Da m Figure 29 Viewing two datasets Note the two tabs at the right hand side of the screen 4 8 Customising the display Switch to a vertical layout 990055 Version 11 39 Oxford Gene Technology ogt To switch from a horizontal to a vertical layout select Tools gt Options gt Display and click Horizontal signal axis Ej d I o n o e E gt o o rm Mu ui gt m L S A m mt GO er pe pE DU Ch m m z D ui m E m dd m a wu S A m e pus o HY L z a E Lai m kes z a o c D 9 Genome gt 252522910004 1 1 Displa Filtered thed 5moo Normalised Raw Sample Information LUZ G3 y 252522910004 1 20 amp ID i gt ED gc Hd WES LZ00 BE qiieoco LE BE i TEM REEL HH ME UE n ec hte MI QWIZOES LZ Ot Chromosome 8 Chromosome Section N XE Rel ED N Lm La rd j N N gt i aom a v See ds 12 2 2 eee SKS 0o rd m Ww m c ei m Ta m 4 N x L SUI 31 dL UI INI ewe E L8L1 rite E i 1080 GLUE i 422 81 awm are iim MES mtu 5 i Whole Genome Figure 30 Vertical display 40 Version 11 990055 ogt Colours To switch the colours of the
9. CytoSure Viewer File View Tools Help Genome Chromosome 5 T 881 LV K FEET ET T Wit UU ug 13 760924Mb Oa EARN qun i a dp nen net s 2108 probes 11 Lr EN l NW NI SL MAI T 1 1 NL mui DEI NEN 1 010 NU LU TEE s Cri du chat B Figure 27 Adjustment of outlier removal values Currently the non filtered data is used for the CBS analysis The ability to analyse filtered data will be available in future CytoSure software versions 4 6 Normalisation and smoothing Normalisation On loading the data into CytoSure the data is automatically normalised by the LOWESS algorithm incorporated into CytoSure It is possible to alter the normalisation window size by Tools gt Options and then clicking on the Tools tab 990055 Version 11 37 ogt Specific normalisation Particularly with custom arrays where a majority of probes may consist of an aberration the LOWESS normalisation may not work correctly There is an option to use specific normalisation In this use the navigation tools to display a probe subset in panel C that will act as control probes for normalisation Select Tools Normalise on the current subset The software will recalculate the normalisation using the probes in the display This feature is unlikely to be required for the analysis of data from Syndrome Plus arrays Smoothing To smooth the array data select Tools gt Smooth Data choose a smoothing
10. All Aberrations Losses NOT autism All Aberrations Losses venn Diagram Latvian NOT Latvian SD a1 T eT I WII II III v EHE EN p A d ay 2009 09 45 29 May Figure 95 Plotting extra groups It is also possible to combine groups To examine the aberration frequency in autistic samples in the Latvian population compared to the aberration frequency of autism in the non Latvian population complete the following Click Combine Existing For Group 1 select Autism For Group 2 select Latvian 990055 Version 11 106 For this example the Combination method will be Intersection And Combination Method k 1 Combination Method Intersection And Union Or Click No to opposite groups and Yes to keep existing groups A group created will be Autism and Latvian A similar procedure of combining existing groups can be used to create a group of Autism and Not Latvian The aberration frequencies are then be displayed on the graph 8 3 Deleting groups Next to the group name is a delete icon which can be clicked to remove a group Groups sue J eee Clickto delete group a autism All Aberrations 58 Gains 24 Losses 34 NOT autism All Aberrations 392 Gains 138 Losses 254 990055 Version 11 107 8 4 Exporting aberration frequency results and statistics To export
11. Il um rm Patient 4 49 m Figure 26 Filtration options The options available are as follows Absolute normalised log ratio allows the user to filter the data so that only data points within a range of log ratio values are included Signal to noise ratio the SNR is calculated and the range of data points that are included can be typed into the box for both green and red signals Signal intensity the signal intensity range that can be included are typed into the box for both the green and red signals These values are going to vary according to which scanner is used However as a guide line for Agilent scanners probes with signals of below 150 and above 50 000 should be excluded Exclude Non Uniform Outliers these are spots which have been flagged as outliers by the Agilent feature extraction software If this box is ticked then these spots are removed from the analysis 990055 Version 11 36 Denoise This is a form of outlier removal in which a percentage of outliers from a set number of probes are removed The default value is 10 outliers from a window of 20 probes So in this case the 2 probes with the highest and 2 probes at the lowest ratios are not shown out of a window of 20 probes The full dataset non de noised is used for any calculations made by CytoSure analysis software To adjust default value click Tools gt Options gt Filters and alter outliers in pop up box
12. AE SlhS l E TSX MlshSA l MNoQ OWSSlsh E EEMloShS AMENS TMEXNloshM l E E E TMEBSBESRRR 15 I 0 1 1 1490963 1 1 EA e 15 2 5 54 eV 8 TPLossihEN 0B 0 8 85 0 1965 e 0 Start PTT N o CEET SU N N tia LIL IMII N p a a lt T x Re run Analysis nm Export Results 1 Whole Genome Figure 73 Output from the loop analysis using the combination method 6 8 Saving aberrations to the database when using combination tool Click on aberration tab If the data had previously been analysed by CBS then these aberrations can be rapidly removed 990055 Version 11 86 T Database management CytoSure analysis software enables aberrations called and annotations added see process described in Chapter 5 to be saved to a database Aberrations saved are recalled on a custom track in the genome view the blue track labelled A see section 4 2 This section describes how to manipulate the database for example to edit or delete data after saving 7 1 Positioning the database file in a location of user s choice The data is stored as a db xml file which by default is located at C Program Files CytoSure Analysis Software This file can be moved using Windows explorer to a different drive e g a shared drive The file should be on a drive that
13. CNV data P CNV data from Shaikh et al Genome Research http genome cshlp org content early 2009 07 10 gr 083501 108 abstract By default this track is turned off To turn on the track see the section on adding back annotation tracks below Having many tracks open can slow the software This extensive study analyzed 2 026 disease free people using an Illumina microarray platform The population was mainly Caucasians or African Americans The data downloaded is the CNV block data please see http cnv chop edu help jsp jsessionid EE8A0897783507053E52A8A2E185C076 sec cnv_view c nv_view for the definition of a CNV block Included when the user hovers over the annotation is the frequency that a particular CNV block is present within the population studied and the number of CNVs within that block which are gains or losses Data is supplied courtesy Center for Biomedical Informatics at the Children s Hospital of Philadelphia http stokes chop edu cbmi 990055 Version 11 23 oft Redon CNV data R CNV data from http www sanger ac uk humgen cnv A2mio By default this track is turned off To turn on the track see the section on adding back annotation tracks below Having many tracks open can slow the software This study looked at 41 samples at very high resolution using multiple arrays with a total of 42million probes Unfortunately there is no gain or loss annotation Data from the ECARUCA database of rare chromo
14. oft Import Annotations from Text File E3 Required Fields Chr Start Stop Mame Begin import From line 100 910164 1145847 IMbClone 128 j 175311915 187408224 Tiepath cl 218 214534 6042798 Mb Clone 263 145434722 146342686 Mb Clone 611 219787705 221890504 Mb Clone 750 90142199 190333427 Mb Clone 01 j 05460369 108512967 IMb Clone 941 354 Figure 21 Adding an annotation track Note that URLs can also be included Click continue Within Custom Annotation Track Details it is possible to assign a name an identifying initial and a colour to each track Once selected the annotation will be visible in the Custom Annotation Table In the genomic view the annotation will also be visible in its own annotation track Custom Annotation Track Details Identifying Initial Colour File Location custom annotation hack Ext Dane Cancel Figure 22 Track details 990055 Version 11 31 Options Classification Report Generation Database Files Protocols Display Genome Normalisation Probe Filtering CBS Annotation Custom Annotation Tracks Import new track Remove selected Edit selected ame Initial Colour File Gename legacy data L custom an Human Annotation Filters Annotation Type Field Copy Number variation v contains L 7 does not contain Create Filter Remove Selected Filter A
15. 06 May 200 X 468919 2524916 2 055 Unclassified 60 3 Variation 33 06 May 200 X 88476955 92191463 3 714 Unclassified 2995 195 Variation 02 TAE 06 May 200 X 468919 3059916 2 59 Unclassified 68 6 Variation 23 06 May 200 7 159218 2973182 2 813 Unclassified 37 1295 Variation 31 I 06 May 200 X 88476955 92191463 3 714 Unclassified 29 1 Variation_02 19 i 20 06 May 200 20 59379047 62263643 2 884 Unclassified 7695 56 Jl Variation 05 06 May 200 4 7751254 8521406 0 77 Unclassified 95 59 Variation 43 gt 12 May 200 1 57709292 67812689 10 103 Unclassified 6 3 Variation_37 27 May 200 X 88583314 92191463 g21 31 q21 32 3 608 de novo p 30 1 0 0 Variation 44 27 May 200 X 164878 2524916 p22 33 2 36 CNY seen i 61 3 0 0 Variation 33 adfasd x 164878 2524916 p22 33 2 36 de novo p 61 3 100 100 Variation 23 adfasd x 88583314 92191463 q21 31 q21 32 3 608 CNY seen i 30 1 0 0 Variation 33 Figure 53 Colour coding according to classification Editing annotating and classifying the aberration list in Aberration view To edit an aberration entry right click on the desired aberration and choose View Aberration This displays the aberration in genomic view To annotate the aberrations see figure 54 select Edit Aberration Details The aberration is classified according to the categories describe
16. Figure 10 Annotation tracks Syndromes S dark blue Some common Syndromic areas have been included Note this is not exhaustive and it is only an indication that this is a Syndromic region Clicking on these bars will take the user to the relevant OMIM entry Genes G light blue Source Ensembl annotation Left click on the bars to link to the relevant Ensembl entry Hight click on the bar to display the following options Link to Ensembl Link to UCSC Link to iHOP information hyperlinked over proteins a network of concurring genes and proteins extending through the scientific literature touching on phenotypes pathologies and gene function Link to Genecards Information about the gene Link to GeneRIF Information about the gene Link to Prospectr Prospectr theoretically calculates if a gene is a disease gene Link to WikiGene Information about the gene 990055 Version 11 21 ih ih e M TR R o N c a of i S 2 49 probes displayed LN I 4i IM Ensembl NN UCSC HOP Patient MEN GeneCards C GeneRIF Prospectr Wiki Gene Figure 11 Links for further information on genes Exons E purple Some genes have the position of the exons included Clicking on these bars will take the user to the relevant Ensembl entry Recombination hotspots surrounded by segmental duplications D yellow Regions of the genome defined as a recombination hotspot by Bail
17. Figure 66 The loop analysis page 6 4 Manually examine replicates Click on each aberration and the aberration will be displayed in genomic view Figure 67 shows an aberration which has been replicated exactly ec Smoothed Display Filtered Raw Normalised o o a X Separation File 1 0 03802084799421313 File 2 0 05282440099787219 File 3 0 019338764032594456 12 7887764 S 0 21 6 a IHR 1I i4 I 7T P 1 ee gu wong pu qu imi ru i iu o Do 2 BH vole lt Ms ation RN Figure 54 An aberration that has replicated exactly There are 3 possible reasons for the aberrations not to be called identically in the replicates The first reason is that the automated aberration detection may not have called the aberration in one dataset This is shown in figure 68 where an aberration has been called in dataset 1 the blue dataset but not in dataset 2 the yellow dataset 990055 Version 11 81 CytoSure Analysis Software File View Tools Help Raw Normalised Smoothed Display Filtered COO S o vert Genome Genomic View Table View Aberrations Loop Analysis 2 Chromosome 5 x Separation File 1 0 03802084799421313 File 2 0 05282440099787219 a4 2 File 3 0 019338764032594456 1 1 i U31 E Ee U i S zm 5 i v d Chr Start Stop Mb i ba 9 i 1 13646742 13649617 0 0 12 H E 1 10 81638193 819438
18. Male Male DO 1D johns DLRSPR INCLUD 02 21 200 21 Fri Jun 19 PK gu Male Male autism O0 1D johns DLRSPR INCLUD 05 23 200 5 Fri Jun 19 P 10184 cg Male Male DO 1D johns DLRSPR INCLUD 07 18 200 4 Fri Jun 19 P 10184 cg Male Male autism DO 1D johns DLRSPR INCLUD 05 09 200 Fri Jun 19 P 10184 cg Male Male DO 1D johns DLRSPR INCLUD 05 09 200 Fri Jun 19 P 10184 cg Male Male DO 1D johns DLRSPR INCLUD 09 02 200 2 Fri Jun 19 P 10184 cg Male Male DO 1D johns DLRSPR INCLUD 09 11 200 3 Fri Jun 19 P 10184 cg Male Male DO 1D johns DLRSPR INCLUD 04 02 200 110 Fri Jun 19 P 10184 cg Male Male DO 1D johns DLRSPR INCLUD 11 05 200 10 Fri Jun 19 P 10184 cg Male Male autism DO 1D johns DLRSPR INCLUD 11 05 200 4 Fri Jun 19 P 10184 cg Male Male DO 1D johns DLRSPR INCLUD 11 05 200 4 Fri Jun 19 P 10184 cg Male Male DO 1D johns DLRSPR INCLUD 11 05 200 4 Fri Jun 19 P 10184 cg Male Male autim DO 1D johns DLRSPR INCLUD 12 02 200 8 Fri Jun 19 P 10184 cg Male Male DO 1D johns DLRSPR INCLUD 11 05 200 4 Fri Jun 19 P 10184 cg Male Male autism DO 1D johns DLRSPR INCLUD 12 02 200 8 Fri Jun 19 P 10184 cg Male Male autism DO 1D johns DLRSPR INCLUD 3 08 i mti Te em 40 MADINA am L
19. W mail ie 16 Feb 200 1 147306825 147644891 0 338 Unclassified 100 100 egrgwer 147306825 147644891 0 338 Unclassified 10095 00 on mens an m Eh el HU K dea rol Sane 1 I95011343 195065925 0 054 Unclassified _ 100 Ld P f un mmm meme ee me DM sample 1 46751472 246928513 0 177 Unclassified 10095 100 cm u ee Fa MEME 17 Feb 200 2 1 3405 3456 oo Unclassified 100 100 m egrawer 2 41201089 241202495 0 0010 Unclassified 100 1 MEME 020 200 9 118497723 18658198 i6 Unclassified o Start 109157762 mL 02 Jan 200 4 18497723 18658198 0 16 Unclassified 0 s Stop 109665904 16 Feb 200 4 18497723 18658198 0 16 Unclassified SE PSAE 9909 18497723 18658198 0 16 Unclassified Size Mb L i i wqegeqwqwe 5 70385760 70622775 0 237 CNY seen i 100 100 areal Unclassified M 05 Feb 200 6 2558676 2660134 0 101 Undessfed fioo ioo 17 Feb 200 7 158815873 H T hoo hoe 17 Feb 2008 775 788 T hioo hoe 02Dec 200 p 7256235 79005 533 Undesfed 95 02 Jan 20 p 256235 79006 533 Undassfied hon ipeo p asezas TUS 0599 Unasfied 100 Rmo s purs VIN psoas ested ju Ge i dau rner 7256235 779NNNA n 533 Unclassified ino ino Genome lt CNY 0 US B Gain Loss CNY CNV Regions f Syndrome Region Important Genes c TF
20. Protocol Mame NEN Create Protocol Load a previously created protocol Protocol Mame v Load Protocol Apply Changes and Clase 990055 Version 11 122 oft Once the user has set all the various settings these settings can be saved as a protocol Type ina protocol name and then click Create Protocol The user can then Load in a previous protocol by selecting the relevant protocol in the drop down menu to restore all the previous settings If during the analysis the user changes any of the settings then the Current Protocol will change to a None 990055 Version 11 123 oft 11 Use of the software in Workflow Mode Routine users can use the software with the settings set as a protocol The principle of this process is that the analysis should be easy to use and can be standardised between multiple users All the settings will be the same However it is still possible for users to change the settings however there will be 2 consequences 1 There will be a warning asking the user if they want to exit the workflow mode 2 In the database and the report the analysis protocol will not be stated There are the following steps when running the software in Workflow mode 1 Select protocol 2 Load 3 Process 4 Describe 5 Review 6 Classify 7 Report 8 End The buttons are across the top of the screen allow the user to move from one step to another 1 Select protocol
21. Various protocols should have been set up using the options highlighted in section 10 Included is the default protocol which contains the OGT settings Click on the Protocol button at the top left of the screen and select a protocol It is important to press Apply Changes and Close button 2 Load 990055 Version 11 124 oft Click on the load button and an option comes up to select either feature extracted files or batch processed files Feature extracted files These should be feature extracted files and so will either be txt in the case of those generated by Agilent feature extraction software or gpr files in the case of GenePix Multiple datasets can be loaded The number depends on the computer and the type of array Typically 3 4 datasets of a 105k array can be loaded Once loaded the dataset will be normalised if appropriate tick box in the Aberration detection is ticked in the protocol The dataset will then be visible for an initial look at the data again if the appropriate tick box in the Aberration detection is ticked in the protocol Batch processed files Feature extracted files can be CBS processed in a batch To batch process feature extraction files click Tools gt Batch processing Then click on the Change button and select the protocol Following the batch processing the files can then be loaded into the workflow using the Load into workflow button Alternatively the processed files can be a
22. 0 H H H HS 7 779058 08 14 2008 Unclassified 0 m dd 08 14 2908 Undassfied pu fm H 77908848 08 14 2008 3 44715974 11 05 2008 3 18436948 09 11 2008 1 147306825 147644891 0 338 Unclassified 100 100 Variation 83 0 05506274 08 14 2008 4 18497723 18658198 Unclassified 100 joo Nanapen G2 Patent P 533263544 12 21 2008 1 Unclassified 100 joo varaton 22 Patient Pati 0 018261 D9 11 2008 Unclassified 100 hioo Variation 45 Paiet 0 03540235 09 11 2008 Unclassified 95 Warshon B7 erea Patet 857247193 08 14 2008 Unclassified 100 Warshon45 berea Patet 8 57247193 08 14 2008 Unclassified 100 Waraton 77 H Paient 857247193 08 14 2008 Unclassified 100 hioo Variation 21 DEFB4 Patet 857247195 08 14 2008 R 7256235 779NNNA n 533 Unclassified NNSA 1n Variatinn 17 DFFR4 Patient 7 A741 75AR INR 14 2NNR v gt 73 Ta Figure 78 The data displayed as aberrations In this case the data has been sorted by clicking on the Chr column heading To view the relevant aberration click on the aberration entry and an image of the aberration in genomic view will be shown 990055 see figure below Version 11 90 Oxford Gene Technology ogt CytoSure Analysis Software File View Tools Help
23. 0 Source file Y WestixDBEMmport SPv2 3 OGT 17 7 2008 z 1 1 txt H 09753520 110058697 1 28 33 1 143659743 143825562 0 16 269 3 Number Features 104969 1 143838567 144137151 0 291 38 mappingFie itest XDB Import SPv2 3 OGT 17 7 2008 z 1 1 txt 45075854 45088496 0 011 26 Chromosome Sect Number of Probes Mapped 103054 40 663683Mb DLE Spread Excellent 0 19623976869998337 Red Signal Quality Excellent Mean 3654 56800051191 Green Signal Quality Excellent Mean 3425 930663437634 Click Here ka Edit Sample Details s lua LIT ET HT mE EL HI In l UE E F LI MHIL TE I 1 E MT T IN E dd BIN A sjam Figure 52 Altering the sample details after File import The annotation list view Once an aberration list has been generated either manually or using automated detection the list can be examined in more detail by clicking on the aberration tab F in figure 1 The aberration view shows a detailed list of the aberrations Each column can be sorted by clicking on the header of the column The annotation provided is shown below RT a ee Start Start of aberration This is automatically entered Stop _ End of aberration This is automatically entered 990055 Version 11 61 Size Sizeof aberration This is automatically entered This is automatically entered Array design Depending on the array used this is automatically en
24. 0 6 16 34331164 34586514 0 25 7 SEED E 17 41543962 41983484 0 43 9 H z x 142618255 142889011 0 27 8 5 70341301 70345683 0 0 26 n Rar ka N Wnt ie ie anal N al LT EB NP GI 4 Ww h Dn n 1 Ou HN DINH d ge ee E Variation CS N NN E d RP11 443K8 Pcenome Mtoe a Tay x N ka II N N Z Figure 69 The aberrations are called with different s 990055 V izes ersion 11 82 In this example if need to alter the dataset 1 blue aberration to the size of the dataset 2 yellow aberration then the first step is to delete the blue aberration by right clicking on the blue block and selecting delete Then right click on the yellow block and select add matching dataset The final reason might be that the automated aberration detection has called a questionable aberration see figure 70 If this is the case then right click on the block and select delete CytoSure Analysis Software e Genomic View Table View Aberrations Loop Analysis Chromosome 22 X Separation File 1 0 03802084799421313 L File 2 0 05262440099787219 File 3 0 019338764032594456 41543962 41963464 0 2616255 142889011 2 41301 70345683 0 0 26 1 2 Figure 70 A questionable aberration 6 5 Re run the loop analysis Once the changes have been made to the aberration lists then rerun the loop analysis as in
25. 98 38 Variation 23 3 27 Feb 200 17 72827808 73072327 0 244 Unclassified 1 1 Variation 5882 a5 27 Feb 200 8 7256229 7789996 0 533 Unclassified 100 100 Variation_45 06 May 200 X 164878 2524916 2 36 Unclassified 61 3 Variation 23 5 06 May 200 X 88476955 92191463 3 714 Unclassified 29 1 Variation_02 un 06 May 200 X 164878 2524916 2 36 Unclassified 6195 395 Variation 23 i 06 May 200 X 88476955 92191463 3 714 Unclassified 29 1 Variation O2 S 06 May 20 X 88476955 92191463 3 714 Unclassified 29 1 Wariation_44 t 9 06 May 200 X 468919 2524916 12 055 Unclassified 60 3 Variation 33 E 06 May 200 21 42665137 45968259 3 303 Unclassified 32 2 Variation 73 06 May 200 5 213686 2670941 2 457 Unclassified 49 38 Variation_44 TI PE 06 May 20 20 59817857 62263643 2 445 Unclassified 8995 67 Variation_35 9 E 06 May 200 11 68333169 70321716 1 988 Unclassified 21 4 Variation 35 06 May 200 7 159218 3202245 3 043 Unclassified 34 11 Variation 31 D T E i4 06 May 200 13 110860140 114026113 3 165 Unclassified 36 3 Variation 30 g 06 May 200 9 94330145 95128662 0 798 Unclassified 0 0 Variation 7754 z 06 May 200 12 120175895 121179066 1 003 Unclassified 36 0 H 3 06 May 200 X 88476955 92191463 3 714 Unclassified 29 1 WVariation_44 i 15 HMM 06 May 200 X 468919 4270976 3 802 Unclassified 62 16 Variation 22
26. Database Files Protocols Display Genome Normalisation E Probe Filtering i CBS Annotation Apply Probe Filters Apply probe filters during protocol Probe Filters Min Max NENT Td Min Red Min Green Signal to Noise Ratio De noise Min Red Max Red Min Green Max Green T Exclude Non Uniform Outliers L D e naising Default Extent of Qublier Removal in De noising 573 Default De naising Window 4 Probes Apply Changes and Close Apply Probe Filters Should the display and the data be filtered See section 4 5 Probe filters Select type of probe filtering see section 4 5 for more details on probe filtering Denoising f denoising is selected then the extent of the denoising can be selected here Denoising removes a certain 96 of probes at either end of the log2 ratio spread in a defined window These metrics can be altered here CBS settings 990055 Version 11 114 Options E Classification Report Generation Protocols Display Genome Normalisation Probe Filtering Aberration Detection Annotation Automatically Process Automatically process using Circular Binary Segmentation during protocol Aberration Calling Options Min Probe Count Min Size Mb Threshold Method Threshold Factor USER DEFINED MEDIAM SEGMENT T Chromosome Average Method Misc Pracess immediately after loading data skip visualisation Number of proces
27. P v Raw Normalised Smoothed Display Filtered DO8 1539 A txt E Invert a a 2 d aud E Le al v Genome Genomic View Table view Aberrations Database Management C Program Files CytoSure Table View Ideogram View Group data by Submission Aberration Commit Aberrations _ Selected Aberration Submission c Size Mb Classification CNY GainjLoss 02 Dec 200 45075972 45083809 0 0070 Unclassified 0 02 3an 200 emen ew pam weed pu 12 Dec 200 45075972 45083809 0070 Undassfied 0 16 Feb 200 45075972 45083809 0 0070 Unclassified o W m eggwe H5075972 5083809 0 0070 Undassfied 0 0 HEP mI mia N inanis A egrgver EM UN NUI TNNT NN 02 Dec 200 109157762 109665904 0 508 Unclassified i ino R L wdefed D IZDec200 f109157762 09665904 0 508 T o i6 Feb 200 1 109157762 09665904 0 508 Undasfed S6 Uh saus IUSI57762 ID9665904 508 Undassfied 56 saus 45732946 aane 0 0090 Undessfed 100 iow 12 Dec 200 1 145375581 145682839 0 907 Undased S 20ec200 147306825 147644891 0 338 CW seeni 0o 02 Jan 20 147306825 147644891 0 338 _ Undesafied 100 4 12 Dec 200 1 147306825 47694891 0 338 Undassfed 79e EH IRI 1 IE N L NE IIT nmm EL AENEID TH LT IMEILIT E Un M L
28. SLOTO1 501 GT 17 7 2008 2 1 1 bt 5A 28878 Loss n SA 1 QGT 17 7 e wN i 30700 Loss n SA 252009010124 SLOTO2 501 OGT 17 7 2008 2 2 bt v 30700 Gan eg 65553 Loss in 5A 08794 Loss in EN 8 901 1 Xin in A Arabs Methuxd Use CHS cas Figure 65 Sample input in loop analysis and selection of the use CBS call analysis method An additional tab will appear Clicking on this tab will display the loop analysis page figure 66 This displays the position of the aberrations in all 3 datasets and will classify the aberrations according to whether they are present in the replicate array results If the aberrations have replicated in the two datasets exactly then the aberration will be classified definitively e g loss in A gain in B However if there are differences between the 2 datasets then aberrations will be classified ambiguously e g Gain in A or Loss in B 990055 Version 11 80 ogt 198277 Lom A Pea o mA HOON n or Los e t lee mH Aw Ganne DOBIT Gun e A o Lone e B OED omn m oe Lom m C NORIO Ion Ao Lowe n T0008 Lee P A o Gun C C dO Lees C v Gm n d 11071 Gan e amp o6 Lo m C 01640044 Gan i B o Cow n C IPM Loos B Gum n Wee el Lows RAG Gam B 19010004 Lows o Gan e C ith Tha Loss P A or Gan m c VWPFAD M Gan m or Lowe m C 398000 Lows m 06 Gan m c IMA Gar C Lo n B 1694 Gun A or Lows n B 1S eO Loss m o6 Gan m C Pel Xat Loss C A or Gan e C
29. aberration that was previously classified as potentially pathogenic Use of the population tab see section 8 display the frequencies of the aberrations in the database The data can then be subdivided on the basis of phenotype and or population for detailed study 990055 Version 11 132 ogt Genomic View Table View Aberrations Database Management Population Analysis Table View Ideogram View Save Aberrations to Database Export Aberration List in Decipher Format Export Aberration Report Dataset 1 252266610008 1 1 Start Stop Cytogene Size Mb Probes Classification CNY Gainj Loss DB CNY DB Gain L CNY Region 16912348 17082258 p36 13 0 169 Unclassified 10095 10095 0 0 Variation 34 E lsurssra beos poki R07 iz hesse 0 eo oe 3 1 I0NSUE 194599 piss 086 i Udasfed S6 De Oh D 1 143732546 143741829 q21 1 0 00900 60 Unclassified 1009 1009 0 9e Variation 23 1 147306825 147644891 q21 1 q21 2 0 338 30 Unclassified 1009 1009 0 0 Variation 34 2 241201089 241202495 q37 3 0 0010 Unclassified 1009 09 0 0 3 56206504 564226045 pl43 0 136 4 Undassfied 0 0 0 Qe o 3 140147820 140147942 q223 0 0 34 Undassified 0 0 Qo Qe o o teases IMSSUS pISSOISGIO16 S7 Undafed O h e o 4 66521191 66643919 q13 2 0 122 Unclassified 9 9 0 9e Wariation_10
30. ac c n s cde Vates um Itt tn c Pr auf am tm quA thm 0 1 Chromosome Section 84 443501 Mb 88 61 8474Mb i 96 968421Mb q21 33 Edit Delete View Region in Ensembl 27 May 2009 11 25 24 05 Jun 2009 12 09 35 05 Jun 2009 12 09 06 06 May 2009 14 24 36 06 May 2009 14 24 09 Figure 49 Selecting the link to SUSPECTS A box will appear with the option of typing in the phenotype SUSPECTS can use a training set of genes if completed however it is not necessary and can be left blank 5 5 Editing and Annotating the Aberration list and saving 5 5 1 Merging aberrations The Segmentation may split an aberration into multiple segments see figure 50 These will be present as multiple entries in the aberration table 990055 Version 11 58 oft Genomic View Table view Aberrations amp Separation File 1 0 034708590955975116 Chromosome 17 Chr Start 17 186719200 17808877 1 08 ptc H Chromosome Section 13 962635Mb 16 883584Mb 18 804534Mb 22 715484HMb 1 I I I I I 1563 probes displayed Figure 50 An aberration spilt into multiple segments It is possible to merge these segments using the following procedure 1 Navigate in the data view to reveal the aberrations to be merged 2 Click Tools Merge Displayed Aberrations see figure 51 3 The aberrations will be presented as a single aberration on the screen and in the aberration table 990055 Vers
31. added to each report Click on the change button and navigate to the file containing the logo The logo can be in a JPEG or BMP file 990055 Version 11 itering Aberration Detection Annotation 119 Report type The user can select the type of report format The HTML format can be opened in Microsoft Word as is therefore editable Report content The Report title will place text at the beginning of the report The Signatory name and position will place text at the end of the report The user can define which information they would like to include in the report If the Notes tab is selected then standard text can then be included in each report More details about the report are discussed in section 5 6 Database settings Options Display Genome Normalisation Probe Filtering Aberration Detection Annotation Classification Report Generation Database Files Protocols Automatically submit Automatically submit results to database during protocol Database Location C Program Files CytoSure Analysis Software db ml Backup Folder Location C Program Files CytoSure Analysis Software m The user can specify whether the aberration results are submitted to the database during the workflow mode 990055 Version 11 120 The location of the database file and the backup file can also be specified Files settings lt Options E Display Genome Normalisation Probe Filtering CES Ann
32. annotation track which show the position of these aberrations The aberration might be coloured red if the aberration is an deletion green if itis a gain and grey if this information is not available Clicking on the Decipher blocks will take the user to the Decipher web site with more information Question 4 Using the right click on the shaded region of an aberration will take the user to the SUSPECTS site This has several tools to help the user decide if any of the genes might be involved in disease Right click on the gene This will bring up some options of sites with publically data on the function of genes See section 4 2 which gives more details on these 990055 Version 11 131 49 probes displayed LN 4i METAM Ensembl anna LIC SC Il IHOP GeneCards GeneRIF Prospectr Wiki Gene T Question 5 The software stores disease causing genes from OMIM and will check the aberration for whether they are present in a particular aberration To determine this then click on the Aberration tab which will open a table of the aberrations Question 6 As the user carries out more experiments the software will save the aberrations and the classifications to a database This data will then be displayed on an annotation track This is track D The aberrations will be coloured according to the classification Therefore normally an aberration which was previously classified as a CNV will coloured a different colour from an
33. are shown with lower brightness The filtration options are discussed below in section 4 5 4 5 Filtration of data Filtration of the data is when certain data points are removed on the basis of certain parameters which are set by the user For example data points which have low signal intensities on the array might be excluded from the analysis The filtration options can be accessed by clicking Tools gt Filters Alternatively they can be accessed using Tools Options Filters A box appears which gives the user various options see figure 26 990055 Version 11 35 oft CytoSure Analysis Software m x File View Tools Help Raw Norm Pus ed Smoothed Display Filtered M D08 1539 4 txt Invert Iv A Inver a Genome Genomic View Table View Aberrations Database Management L E Chromosome 9 X Separation i1 17 T TT File 1 0 0155695084972105 TM a ijj 1 d E i Chr Start Stop Mb B o Filters 5 i 21 Absolute Normalised Log Ratio Chromosome Section T E Signal to Noise Ratio EE 0 0Mb B D F B 2 M Min Red Max Red Min Green Max Green Signal Intensity 3 2 1 000 50 000 1 000 so 000 S 1 E Exclude Non Uniform Outliers TE i H i 3 L ELL I INE gl T mis ll il TT tH MEL UI LIE MEI ll 1 0 AE RN IN lll l LOIU IUN n a I nanna 4 ae
34. areas Aberrations will be placed in the Aberration window where they can be edited if required 9 Itis recommended that once the CBS has run the file is saved using File gt Save The CBS results are saved 10 If the threshold values need to bealtered there is no need to rerun the CBS Select Tools Options Selection of the Tools tab and the CBS option changes the threshold values see figure 35 po a Genomic view IUE Global Normalisation Window Size Probes 5 000 Signal Processing DAC Default Signal Processing Window Probes Circular Binary Segmentation Click Here For CBS Options APEWA E l Ee l ll l ad en uu n Figure 43 Changing the CBS threshold values 990055 Version 11 52 5 2 Batching automated aberration detection The software enables multiple files to be segmented using the CBS in a batch process This means that the software can be left unattended to process the data CytoSure Analysis Software Sele File View MEE Help Raw Mormalised Smoothed Display Filtered R o al nerrations Database Management BEH da E E K It Batch Processing Options I A Chromosome Section Na 1 mau HI I kal Eh l A LH ka l a Hi m m BEHINI MIN m d N EL N n E G en Figure 44 Starting batch processing 1 In Tools click on Batch processing see figure 44 2 A dialog box opens figure 45 Complete the foll
35. core and multiple threads are used the processing time should typically be 4 to 5 minutes for Syndrome Plus 2x105k data CytoSure software works well on a non quad computer however the automated aberration detection running time will be slower To install the software double click on the file CytoSure Analysis software exe to run the installer and follow the on screen instructions To update CytoSure analysis software uninstall the old version by selecting Start gt All Programs gt CytoSure Interpret software gt Uninstall Next install the most recent version as described above 2 Running CytoSure Interpret software Start the application via the Windows Start Menu This will most likely be done by clicking Start gt All Programs gt CytoSure analysis software gt CytoSure analysis software 990055 Version 11 7 Oxford Gene Technology Gerome View Table View Mer gera Database Management Popustion Analyses Sele L cd Cuomosome Figure 1 Opening interface The opening interface is displayed in figure 1 There are six parts to the interface 990055 Version 11 8 A Chromosome selection panel This is used to select the chromosome whose data are displayed in the chromosome overview and the chromosome section view lt also offers the option to display data for the whole genome B Chromosome overview This panel displays a summary of the dataset selected in the chromosome selection panel including a
36. or whole genome view 990055 Version 11 127 Oxford Gene Technology Genome un LP en Gu IP c Lim C P un 1 LET ss iD A LE E LR N N P Lr E N N eal Loo 1 JJ lt i Whole Genome 990055 Genomic View Table View Aberrations Database Management Population Analysis Chromosome 3 E LE ELLO aaeeio AJ MULA OL IL ALBNECL oe ee ee Sm V Spr en ES RU H dT T 2 1 Chromosome Section 163 524658Mb 163 752841Mb 163 981025Mb 164 209208Mb 3 2 vL Variatio RP11 498P 15 BL Patient 2002 al s 1811 amp Patient 1989 atient 1561 Locate the aberration Version 11 Raw Normalised Smoothed Display Filtered Aberrations Chr Start Remove C T T tooor6zzszsz ER S amp 3 Z Y f000I622s2s2Z L x E ogt 128 b Zoom in All probes shown on the screen should be within the aberration There should be no flanking probes visible otherwise these will be included in the export E 0 t 1 1 Experiment Semple M vs Reference M Genome a TIE Li L r 1 t3 D LI FE U 19 H S 11 L L 3 2 u m B 1 Ch Start Stop Mo a7 i r D 420 10 probes displayed Phenotype Details sez ee N Variation 4361 Patient 1989 Expand so only the data points in the a
37. right click on the aberration and the various classifications will come up Select the relevant aberration Further information can be added to aberration table such as in the Notes page then text can be typed in Report Once the classification is complete then clicking on the report button will carry out the following processes 1 Save the aberrations to the database 2 Produce the report in the format specified by the protocol 3 Save the classification and annotation as a cgh file This can be opened using File gt Open 990055 Version 11 133 Both the report and the database will contain a record of which protocol was used to carry out the analysis Finish Clicking on the Finish button will complete the process and the software will now be ready to load new datasets The user can click on the Finish button and the dataset will be closed following a warning Altering the protocol in the middle of workflow mode Changing the protocol in the middle of workflow mode or altering the settings will result in the software producing a warning The data can still be analysed using the revised settings however the report and the database will not contain a record of an analysis protocol used 12 Other features Array metrics X chromosome separation is shown at the top right hand corner of the chromosome section view C It calculates the mean ratio of the Chromosome X probes It is intended for experiments with male and f
38. the aberration frequency results complete the following As in the Genome view navigate so that the region of interest is present in the viewer Click Add to add that region to the list Options amp All Aberrations C 7 Gains C 7 Losses Groups Regions venn Diagram Bin size pixels Regions Start Stop z 85656423 19911467 24 91022276 R 9 U meines 1 venn Diagram autism NOT autism Figure 96 Adding regions to the table To delete a region from the list highlight the region to delete and click Remove The software incorporates some simple statistics for determining the confidence value of an aberration frequency result There are two statistical tests both of which will test if there is a significant difference between two groups The tests are 990055 Version 11 108 Chi squared test Fisher exact test 2 tailed A Fisher exact test will be used if in the contingency table an expected value is below 10 otherwise a Chi squared test will be used Click Export and a txt file will be generated with the following metrics Chromosomal position of selected genomic region Start position of selected genomic region Stop position of selected genomic region Number of aberrations in the selected genomic region Number of losses in the selected genomic region Number of gains in the selected genomic region Mean aberration frequency in the selected genomic region Mean lo
39. there can be several terms included in this field e g autism developmental delay etc 990055 Version 11 101 Genomic View Table View Aberrations Database Management Population Analysis Figure 91 Adding sample details to the database C Program FilesCytoSu Table View Ideogram View QC Trends Group data by Submission Aberration Refresh Submissions Scan Date b vy Sub v Fileloca ArrayBa Arra Sam v Sam v Refe Sam v Phenotype Details v boo vr IHE ve SIMI Gases nae ULliTUU cUU Je PEE SUE 12 eee PULUT CY aC aC VOTLO s Y Jot gt LLP JP A o SLL s a a E 01 09 200 10 Fri Jun 19 P 10184 cg Male Male autism DO 1D johns DLRSPR INCLUD 01 09 200 2 Fri Jun 19 P 10184 cg Male Male DO 1D johns DLRSPR INCLUD 01 09 200 4 Fri Jun 19 P 10184 cg Male Male DO 1D johns DLRSPR INCLUD 01 09 200 2 Fri Jun 19 P 10184 cg T Male Male autism DO 1D johns DLRSPR INCLUD 01 14 200 1 Fri Jun 19 P 10184 cg Male DO 1D johns DLRSPR INCLUD 01 14 200 1 P 10184 cg johns DLRSPR INCLUD 01 14 200 2 P 10184 cg autism DO 1D johns DLRSPR INCLUD 01 14 200 2 Fri Jun 19
40. visible in figure 48 5 8 P value 1 The method of calculating the P value involves working out the difference in means between the probes in the aberrations which have been highlighted by the user and the mean in the control region This value is then divided by the standard deviation of the control region The P value is then calculated assuming a normal distribution 2 The control region comprises all probes in the same chromosome that have not been highlighted as an aberration by the user 3 Therefore it is important to highlight all the aberrations in the chromosome to ensure that the control region is correct 4 There are many different statistical tests that can be carried out The test used here is a conservative test it does not take account of the number of probes in the aberration The P value can be used as an indication of the quality of the result However it is important not to place too much emphasis on the P value and also consider the number of probes in the aberration Other tests such as the t test or Mann Whitney test frequently give much lower P values on the same datasets If there are two datasets e g dye swap the software uses both datasets to calculate the P value 990055 Version 11 75 6 Loop experimental design An alternative experimental design to the reference design is the loop design This uses 3 arrays for 3 samples and results in duplicate data for each sample Hybridizations are carried out a
41. 11 117 Classification settings Options Normalisation Probe Filtering CBS Annotation Report Generation Database Files Protocols Add new classification Mame Colour du Add Classification Remove Selected Existing classifications Colour Apply Changes and Close In the aberration table see section 5 5 2 it is possible to classify the aberration such as CNV de novo etc In this section the user can define their own classification terms and what colour the annotation blocks should be Reporting settings This section outlines the customerisation of the report 990055 Version 11 118 Options Display Genome Classification Report Generation Database Files Protocols Automatically generate report Logo Automatically generate report during protocol Report Type HTML G PDF Tab Delimited Text Report Content Include ideogram in report Always generate new images Report Title Cytosure Analvsis Software Aberration Report Signatory Mame Signatory Position PDF Image Format Ce PNG Medium Quality C SVG High Quality Sample Info Aberration Info Summary Columns Motes Template Excluded Included Submission Date gt p ArayBar coe 00000 Automatically generate report during protocol If ticked then in workflow mode the software will automatically generate a report after the user has annotated the aberratons Logo A logo can be
42. 11512 saat g Source File post RT_252266610008_S01_H_ t AF Design Type 15k Aneuploidy i 1 ZH E de Se t 4 M i FA lf Mapped Probes 15164 15744 Pei x L M us o 2 z or er r x caf k TENA a 1 L xe r A A a ML AP 5 d Voss w 4 ANNA t K 5 BYAPRUI i 8 3 TE HE RE i E I _ SO IB PUE NNI MUN ND WM TERUEL EIL THRILLER UT ET EHI LIEN LET TENET UH NE 1 DNI UNT DIEI TI p T T HR ix jv EXISTE b ii EED MD D D gt l i Aberrations Chr Start Stop Mb o Uo Ma Onn m met oy a n On STU LI IHI 1 lN 1 N 1 1 TUT TET Ru y A NT ELE 1 I nome CALENTAMIENTO TE INL MT TUI MUI Remove TO mm auc ler B Figure 6 The viewer with feature extracted files loaded 4 1 Navigation The chromosome selection panel A displays an icon for each chromosome in the Human Genome This allows the user to view the data for a specific chromosome At the bottom of the 990055 Version 11 15 oft panel is a button entitled Whole Genome this allows the user to view all the data available To view a specific chromosome dataset click its corresponding button A chromosome selection button Click on this button to display data for chromosome 10 The M button refers to Mitochondrial DNA i dine Click on this button to display data for the whole genome L
43. 4 Variatio haet 00557175 Unclassified 43 Wariation_22960 Variation 2860 Variation 31719 Varia OE C a CC TRI Unclassified 162 Variation 1396 Variation 1354 Variation 2528 variatio gt desfel oe OO aa Taga a LSR r devel oe UE t 0 S y s r esfel e T a a S O O s Unclassified 100 100 Variation 7028 Variation 37621 Variation 30648 Variat 58888 Unclassified 100 Variation 0376 Variation 34656 Variation 31963 Variat Ha Unclassified 100 Pariation 7028 Variation 0376 Variation 8235 Variation o I InclazciFiad 1n Tine Mariskinn FOR l ari abin GOA Waristinn Tale Waristi 0 17116927F Figure 71 Removing aberrations so only the loop data remains 6 7 Alternative method the combination method An alternative method to carrying out a loop analysis is to combine the dataset prior to doing the analysis To do this click Tools gt Loop analysis The loop analysis box opens A dialog box will appear that will allow the user to input the sample details and to specify with which dye the samples were labelled If some of the sample details were inputted during file input then some of these details will be automatically generated Under analysis method click Combine datasets step 1 in figure 72 then set the Threshold step 2 in figure 72 and the minimum number of probes step 3 in figure 72 990055 Version 11 84 ost CytaSure Analysis Softw
44. 4 1Mb Clone Array 190333427 1Mb Clone Array 108512967 1Mb Clone Array 146342686 1Mb Clone Array 7843532 1Mb Clone Array 146342686 1Mb Clone Array 218866017 1Mb Clone Array 224229492 Tilepath cloneset 146342686 1Mb Clone Array 146204124 Tilepath cloneset 246181016 1Mb Clone Array 111090422 1Mb Clone Array 225807728 1Mb Clone Array 121186957 WTSI TPA 23311475 1Mb Clone Array 19752047 Cytochip V1 1 146375718 Agilent Human 244 70814713 1Mb Clone Array Click Import New track and navigate to the txt file The box below opens 990055 Version 11 29 ogt import Annotations from Text File Required Fields Chr Start Stop Mame Begin import Fram line E B3 g25 927 941 182551565 groz Cykochip v1 1 14480 7807 456375718 Agilent Hu BOB S2 70814713 1Mb Clone A 70717489 70814713 1Mb Clone A Figure 20 Adding an annotation track E Number Chromosome 218 1o ess 6042798 HR Clone A 611 750 i 190192199 190333427 Mb Clone A asi 1 Rn Mb Cone A Mb Clone A The user needs to input where the data begins In this case the data starts at Line 2 as the first line contains the headers Next the user needs to define the type of data present in each column To do this select the appropriate option from the drop down menu at the top of each column In this case the first column is Name The second column is Chr the third column is Start and so on 990055 Version 11 30
45. 55 Options Display Genome Normalisation Probe Filtering Aberration Detection Annotation Classification Report Generation Database Files Protocols Automatically generate report Logo Automatically generate report during protocol Report Type CO HTML Ge PDF Tab Delimited Text Report Content Include ideogram in report Report Title Cytosure Analysis Software Aberration Report Signatory Mame Signatory Position Sample Info Aberration Info Summary Columns Notes Template Excluded Included Motes Reference Gender Sample Details henotype Details QC Metrics CBS Calling Settings Apply Changes and Close Figure 61 Customizing the report The HTML and pdf reports have a similar format The HTML files can be opened in Microsoft Word for further editing A logo can be added to the report by selecting the change button and by navigating to a file containing the relevant logo The files must be in JPEG or BMP format The information included in the Sample Info Aberration Info and Summary columns are all user definable Information can be added or removed from the report by highlighting the relevant information and selecting the or Em button The Notes Template allows the user to add relevant text which will appear on all the reports Version 11 72 ogt The layout of the report is shown in the Figures below 12312312 12312312 Scan Oste 04 17 2009 17 1947 ID LIEU Thu
46. 64 0 3 23 do 10 Chromosome Section 12 7887764 8099181 0 21 6 x it 70 336919Mb 70 341301Mb 70 345683Mb 70 350065Mb 14 i o 6 ooo Mohd e I o ce LJ o 218 o eA eae Ci e B Hen a I a 20 pd M se 1 s iY aO O ODS i Whole i Variation 4452 fs Genome ci P171 172 173 add J Remove J Ext Figure 68 One dataset is not called If the user wants to call the yellow dataset as the blue dataset then in this example right click on the blue block and select add matching aberration ogt A second reason for differences in the replication is due to different sizes of aberration called figure 69 CytoSure Analysis Software File View Tools Help quR M S Raw Normalised Smoothed Display Filtered o E Genome Genomic View Table View Aberrations Loop Analysis JE ka N J R T n LES 4 E x Separation File 1 0 03802084799421313 File 2 0 05282440099787219 File 3 0 019338764032594456 SGE L Chr Start Stop Mb 1 1 16703542 8 S 1 45448492 45680646 0 23 7 a 10 Chromosome Section 2 108529680 108862771 0 33 9 H E 6 32079821 32082830 0 0 12 108 038818Mb 108 3891 25Mb 108 739432Mb 109 089739Mb ol recul xs cal 13 99434951 99436362 0 0 8 z 15 18810004 18848272 0 03 14 213 g 14 15 19464734 19465583 0
47. 73432 ls 1 147305825 47644801 0 358 dassified 100 100 Wariation_23 Patiert Pati 0 01113933 Loss 15 19353031 19432994 OON dessified 100 100 Variction_34 Potiert Pati 00486501 Figure 86 Highlight the submission of the data to be sorted 2 To filter the data Next to the column header that is to be filtered there is a size icon REL Click on this icon and a dialog box will appear Edit Size Mb filters Figure 87 Filter options 990055 Version 11 97 Type in the parameters of the data that should be shown For example if the user wants to visualise only aberrations of a size between 0 1 and 0 5Mb type 0 1 and 0 5 in the Between boxes and click Apply Size To remove the filtration click on the icon when the dialog box appears press clear To sort the data click on the column title A new icon will appear _ Zeno alongside Click onthe to sort the data in ascending order The icon will change toa andthe data can be sorted in descending order 7 6 Displaying QC metrics QC metrics of data stored in the database can now be tracked over time Navigate to the database modification page Highlight the relevant submissions by holding the shift key and clicking on the submissions see figure 88 below Click on the QC Trends button circled in figure 88 below Genomic View Table View Aberrations Database Management Prog
48. 8 11 9256235 7790006 p231 0 533 7 Undassfied 1007 1009 0 9e Variation 27 B 9uSuS SMESI pIZS OU 4 Undasfed 10 100e D Oh Varton 27 8 loss 7062586 ibi 0 0010 5 hesse oe x 8 bas biem Wia Wie W jundessfed 6590 free ore aration 3 S090 784666 p43 ozs 8 Udassfied 10 100 0 o iana B6 9 2627345 2728775 p242 0101 Unclassified 20 0 0 O o o o M a n C NN o 43598672 43599699 pl2 0000 6 Udassfed 100 100 0 o aana 28 Hestes isores nli Dus s ested o O o o Dh P 137303087 137437381 090 3 0 134 4 Unclassified 100 100 0 0 Wariation_34 sos S904676 piil OOA 6 T O Waitin 6 ieiet 16196162 pisi oie ndesfed BP 0 x p c ee a a ISSIS7IS GNO aie 1 196 Undasfed 100 00 Qe e Variation 30 15 1171815 21190973 qil 22 0 019 8 Undassfied 1009 1009 0 9e Variation 30 15 8144735 28294783 q13 2 0 15 4 Undassfied 1007 1009 0 9e Variation 87 75 ues WUxMn ql 0447 Unease 17 We Oe D ain 3b 16 CINES 1103415 013 3 E 141 al E oes Jo 2 x Variation 53 7 lt gt Notes One of these columns contains information about if the aberration contains any important genes Classification With these tools it should be possible to classify each aberration To do this click on aberration tab then
49. 80909 13pbradyO X 2000 MN 90796 ERa X Awv2000 MN TRT Kra X Aw 2009 OW 909 8 ERa X Xwv2000 05 23546 raa 3X Jw 2009 MB MISTS I pbradyo X Jwv2000 kv 201758 rda X Jerr 2009 x 115531 6 pbr adyO x Sarr 2009 iw 265472 20 pir adyO x x Jarr 2009 Ds 377977 20 p r adyO x Jarre 2009 pert 340527 19 gbr sdo x Jarr 2009 i 13 DoudgasH PMexryo eM 13DoueHM X40109 gt Jarry 2009 1 Dougash Y10184 g betes Lt e as I T9 e e 37 Ner MSS ew vv s Derr abons Or Rat Rep See Mb Orka b 290629 30107197 0 61 1 ZO 2099 1 16 Automation Lewel Manos jextra commerts can be added L Orged s Figure 85 Image showing the area where changes to the annotation can be made 3 Below the image is a scroll bar where changes to the annotation Classification and Comments can be made If the window cannot be seen it might be necessary to fully maximise CytoSure Analysis software window Display Filtered aln 4 Click Apply changes to save the changes to the database 5 In order to edit the aberration click the commit button at the top right of screen see figure 60 990055 Version 11 ogt 7 5 Sorting or Filtration of data in the internal database lt is possible in CytoSure analysis software to filter aberrations that are stored within the database For example a user might want to only see aberrations of
50. 82 O 18654144 CGH DII j4 asea3482 3SM3MIA 1579 18310 021362061 2 1651065 CGH ony ia 35730688 3STNUZTA 3596 0 2998 0 026239263 027118334 i m En JERS LR SU gun mes R E 12T j Ei 15 umm j j GH 0117 35650463 35650522 6944 0 7275 0 0 06718030 0 0389155 GH 0117 35615037 356150906 4027 0 4572 0 0 18311993 0 1548975 EHNI N z SM N kek imi ha N Figure 36 Table view showing detailed probe information 4 10 Aberration tab and ideogram view To access the table of aberrations click on the aberration tab F on figure 1 This can be used to annotate those aberrations which have been generated either by automated aberration detection section 5 1 or manually section 5 2 See section 5 3 for more details To view aberrations on an ideogram click on the radio button figure 37 The gains are indicated in green and the losses in red see Fig 38 990055 Version 11 45 Oxford Gene Technology ogt CytoSure Analysis Software Figure 38 The ideogram view Click on an individual chromosome to view full screen Re click on the chromosome to return to the standard ideogram view 990055 Version 11 46 ogt 4 11 Aneuploidy testing This feature rapidly tests the presence of aneuploidies in the sample
51. 95970 91680411 i Unclassified 1 30 Congenital d ka 16 32270938 32862289 0 591 Unclassified 91955 Varii PPatentPati 0 00673482 1 147306825 147644891 0 338 Unclassified 799 o ywenionS3 Patent Pati 0 01117044 1 109157762 109665904 oso Umdmsed Du 0 Raman 8 7256235 7790006 0 533 Unclassified 10050 Wars 27 EBA Patent 857247193 Figure 94 Adding extra sample details to the database Now the samples are classified as Autism or not Phenotype Details and Latvia or not Sample Details Following on from the previous example Click Add New Group Select Sample details Type in Latvia and click Apply Ss ee ug Details filters C anbains Clear 990055 Latvia Apply x Latvia O 00000000 Version 11 Oxford Gene Technology Name the group Latvian Add the opposite group e g NOT Latvian There are now 4 groups Autism No Autism Latvian No Latvian Note The Venn diagram will show a maximum of 3 groups the first 3 checked Genomic View Table View Aberrations Database Management Population Analysis Chromosome X Options m All Aberrations O Gains O Losses Pixels per sample Groups Chromosome Section Combine existing m e lii 86 753058Mb 88 680817Mb E 90 608577Mb 92 3336337Mb
52. Aberration b 31 577825Mb 33 517025Mb a re you sure you want Eo delete this aberration me This action is irreversible Chr Start 1525056556 30517325 Ae T LINGO 17 3 l l II l l l R BEN H H NELLE I Figure 83 Deleting an aberration in the database 990055 Version 11 94 Oxford Gene Technology ogt CytoSure Analysis Software v 068 302 5077 _1 2 00t wd Irryert Ray Normalised moothed Genome Genomic c View Table View Aberrations Database Management r PEE C Program Fies Cyt Group data by O Submission L rt M A A N M Fe A M M M M M M e ae e M e A l M M m M a A A 18497723 18658198 0 16 LAZ tag if v Figure 84 Committing changes to the database 7 4 Editing aberrations in the internal database In order to edit the annotation associated with an aberration within the database the following process is used 1 Navigate to the aberration as described in section 7 1 2 Click on the relevant aberration An image of the aberration should appear see figure below 990055 Version 11 95 Program FlesiC ytoSure Analysis Group data by Submission Aberration Refresh dbeesionis Selected Aber ation Date Sample D Abert User Jav2009 0 3
53. Aug 27 1324323 OST 2009 pot 17 RT 252200010008 0 M CGM we 9 FeoQ 2 no norm tot CULTE M 2 2200010008 7 S CytoSure Analysis Software Aberration Report for Sample ID Pte HU 1 con cos I 1 Sarnpee 10 12212212 Samp Gender EE lt o HI 2ge 1M DNA for 21 Juneco a E EC E Phenotype amp Phenotype 22 GE 0 ll mm Sample info C TlIale lalalal 2 lala PBB ele Tele lalallala H n z L x n n PIEITBIEIS minj IiRIBIN N NM I ja ti pa m L my 2 B z z n E H EL ELT BILAL EL aS ist S l e bI aI Miele lal ele lade pela ele lal a on Ideogram m n x K x Y n D v i x L eee oe eee eee 1 ee ee oe ee eee ee ee ee B LB LR LB LN lB lU lE LS LS eee 990055 Version 11 73 ogt 12312312 Summary columns Cytogenetic Location Chr q21 31 q21 32 12312312 o At 7 da e ij WM i t Aberration info 74 Version 11 990055 Jane Smith 4 Signatory amp position Lab Head Page of 7 Figure 62 A custom report 5 7 Exporting the aberration results in Decipher format When the list of aberrations has been generated and edited it can also be saved in a format that is ready to be uploaded to Decipher simply by selecting the button Export the Aberration List in Decipher Format
54. CytoSure software the CBS can be run for each dataset Please see section 5 1 for more details 6 2 Check the aberrations manually The process of manually checking the aberrations is easier to do if 1 dataset is checked at a time Alterations to the list of aberrations can be done using the manual aberration tools see section 5 2 It is necessary to use the tabs on the aberration table to toggle between the datasets see figure 63 990055 Version 11 77 Oxford Gene Technology Dataset 1 Dataset 2 Dataset 3 Ganom View Table View Aberrations Chromosome 2 Chromosome Section 108 34960 o mom 109 1266658 gt 18677217 lant esa Ub 199598158 4 o Figure 63 Checking the automated calling 6 3 Running the loop analysis Click Tools gt Run analysis 990055 Version 11 78 CytoSure Analysis Software File View ECES Help Export Aberration Report 2 1 _2 txt E Invert aerrations Mormalise on current subset Identify Aberrations 2 1 2 txt E Invert De noise Raw No desk sed Smooth Pees 1 gt IM ogt Smoothed Display Filtered a ka UL ET MIELE 17 WE W TT alle Nr Mm W m LINE UL EI TT 3 9 I TA ET 1 H L Merge Displayed Aberrations Chromosome Section 0 0Mb 82 416573Mb 164 833146Mb 247 249719Mb miu un Un UE oom en c IINE N GD 25610 probes displayed HE Sami P
55. IE REI 1 31 kel G A disabled chromosome selection button There are no probes mapped to this chromosome in the current dataset S The chromosome overview B displays a summary of the dataset selected in the chromosome selection panel A If the data has been smoothed this component also shows a line indicating the smoothed values of the displayed data Initially the scatter plot is outlined with a dashed rectangle indicating the current section of the chromosome or genome that is displayed in the chromosome section view C The user can select a subsection of the chromosomal genomic data by dragging the mouse between the required start and stop base positions of the chromosomal genomic data The chromosome section view C will then be updated accordingly If the mouse is positioned within the chromosome overview B the user is also able to scroll through the chromosome genome whilst maintaining the size of the selection window by using the mouse wheel or the arrow keys Chromosome section view C displays the data subset selected in the chromosome overview B as a scatter plot of probe location against loge red green mean signal ratio The user is able to view information about each displayed probe by hovering over the probe with the mouse 990055 Version 11 16 File View Tools Help Genome Genomic View Table View Aberrations Database Management Population Analysis Sample Information K
56. In genomic view click Tools gt Aneuploidy Summary This provides a rapid method for averaging all probes on each chromosome and plotting a box and whisker plot The plot shows the following position of the mean median 50 percentile lower quartile 25 percentile upper quartile 75 percentile maximum value minimum value oee figure 39 for details Zoom into regions on the plot by dragging the mouse icon over the relevant region Genomic View Table View Aberrations Database Management Chromosome 1 dou Ph pm t agp Re eH e pe igit U U U U U U D Forme e S n V U U U U U U Chromosome Summary Chromosome Section 245 400322Mb 240 10005Mb Normalised Log Ratio 1 2 3 4 6 H H B 1B 10 12 13 14 18 18 17 1H 18 20 21 22 X Y Chromosome B Dataset 1 Figure 39 Diagram of the Aneuploidy Summary screen 990055 Version 11 47 oft Maximum value e s Upper quartile 75 percentile Median the line Mean the dot 3 ai S 2x 50 percentile Lower percentile 25 percentile Minimum value Figure 40 Diagram explaining the different values on the box and whisker plot 5 Adding aberrations to generate an aberration list annotating saving and exporting for printing 5 1 Automated aberration detection The program uses a modified form of Circular Binary Segmentation CBS to divide the data into regions
57. Jam 200 aso75972 48089809 0 0070 Unclassified oe 0o Oo o me araz 08 14 2908 12 Dec 200 1 45075972 aso83809 0 0070 Unclassified 0 re H PM is Feb 200 o deoyssz 45063609 0 0070 __Undlassfied om be OO oO PIQ amp OO 8711736 2 D8 14 2008 eqrwer i SS MSOSGSDOS 0 0070 JUndassfed o ba 2 O 13408860 08 14 2008 ewower 103962497 104105948 143 Undessfed 100 hox Waihonz0 o T 9 142008 Q Dec20D 09IS7762 0966994 0 508 Undesfed Qu O O O O To oono D6 14 2008 mm ooo a dossz7e2 queeese 0 508 Qundasfed om Ooo Too o 8100987 D8 14 2008 o Bj recze asesan pum Wasted 0 ooo ooo o 9 81903070 98 14 2908 Nd sran Y mses asesan sos Qundesfed Res Uk O Tooo To oome eaeoe O CHEMENE e o oors Qos esoo4 H Bu 0 Too o 9 986112 D8 14 2008 s exe h isrusesd 149741829 0 0090 Unclassified 100 hoos Variation a0 Potent Pati 0 03946891 08 14 2008 GEN 12 Dec 200 oo ssess 145682839 0 307 Undessfed 5 Marishon 3 Trombost Patient Pati 0 95971019 08 14 2008 eT 50 26 denoems 147644891 ass NY seeni 100 Veriston S3 Patient Pati 01113995 08 14 2008 Bls unico 1 2068s 147644891 398 undassfied 100 Wariation_34 Patient Pati D 0L113993 D8 14 2008
58. Open The sample details box appears figure 3 Select which dye the sample was labelled with The software can then calculate whether an aberration is a gain or a loss In addition select the sex of both the sample and the reference Sample details and phenotype details can also be entered It may also be necessary to select the array type this will depend on array type used Click on the continue box File View Tools Help PARE Details N gt Load e Sample Label Cy3 Ocys ER RR E Display Sample Gender aw Normalised Smoothed Filtered Male Female e o E Sample ID Genome Genomic View Table View A Selected Chromosome brmation Sample Details t Sample M vs Reference F etails Chromosome Section Phenotype Details e Details tart Stop Mb 2 b L Whole f Genome Add Figure 3 Sample details box 990055 Version 11 ogt The software displays a normalisation box see figure 4 CytoSure software incorporates a LOWESS normalisation algorithm However some feature extraction software e g Agilent feature extraction incorporates a normalisation option too Therefore if the data has already been normalised then click No The recommended method is that the feature extracted data has not been normalised see OGT technical note and the user clicks Yes to featur
59. RU eee TTT 39 4 8 Customising tne diS play PETIT Ut I m TET 39 4 9 Table view Ao ERN T UT m 44 4 10 Aberration tab and ideogram VIEW cccccecece eee 45 ATi Y Ee eie 1e RETE 47 5 Adding aberrations to generate an aberration list annotating saving and lt gt dele g t rc eee ee eer ee eee errs 48 5 1 Automated aberration Aetection 0c cece cece eee ee eee eee eeaee cease eenaeeess 48 5 2 Batching automated aberration detection eeeeeueuuuuueeues 53 Duo Manual aberration ete OM essa dauid ekact ens EE prs dupPEQuSy IM M Pas nd Site E PER oer bdUS 55 5 4 ES a ns cet UV E E E UNDO EEE 57 5 5 Editing and annotating the aberration list and saving 60 5 5 1 Customising the classification terms 5 6 Exporting the aberration results for Printing cccc e eee 70 5 6 LCustOmiSing INS repor a anahevncnusdortncansarcasevestbenecenseucenetneese 73 5 7 Exporting the aberration results in Decipher format 75 5 8 uas aces tiie ore ware A e ENET 75 6 Loop experimental deS ssepius rR 76 6 1 Import the 3 arrays and run the CBS cccsccesseeeceecsseesesenesesentens T7 990055 Version 11 2 9 10 11 12 13 990055 6 2 Checking the aberrations manually eee e e e e cece eee e sees eeeeeeeeeeeaeeeanees 56 6 3 Running the loop e oan eee TOLL OST 57 6 4 Manua
60. T LT PM Oo n T PEE nr PNPPP TRIALS Aberrations Chr v Start Stop Cytogene Size v Classif CMY v Gain L DB CNY DB GainjL CNY Regions Syndr Impor v AlGe 7 31461588 31504608 E 0 043 Uncl 7 38 zi 100 100 USR 0 Variation 31 163997228 164101835 q26 1 0 104 Unclassified 100 noo o e aR 246805521 46862088 g 4 amp 056 Undassfied 1009 L fom 09 jWarabonGS ORTILO 32146491 32203395 p133 amp 056 Undassfed 100 o 0 bo T aose 32558677 32660134 p21 32 101 Unclassified 100 1005 o f Variation 32 HLADRES 42014069 M2691944 pi2 0 677 Unclassified 1009 1005 o 9o variation O7 RP11 146D1 5186463 qi3 1 0 021 jundassfed o Qo X b SPA RELA SPAI 028601 p223 0 053 jUndassfed o o Qe 0 30 0 CPW 9490689 q11 1 q11 2 0 866 Unclassified 100 1007 9 0 Variation 34 OR4K2 ALS 5529608 p36 11 0 083 Undassfed 100 1007 9 9b Variation 68 RHD Clorf63 RH 238122 p13 3 o0050 Unclassified 100 0 Qn o 3 lt 140518817 154582473 27 2328 14 063 Unda
61. X 4 x p lt B Decipher Regions v Amy ue U U 4 U U a 4 fs Figure 79 Detailed image of an aberration stored in the database Viewing the data within the database as an ideogram To display the data in the database as an ideogram click on the relevant radio button as shown in figure 80 This will display the data as an ideogram figure 81 In this view the green bars represent gains whilst the red bars represent deletions 990055 Version 11 91 Oxford Gene Technology ren VW Tiia ume Dean OAA Le H Crogan ecr ehr O Td tn Orn m re date O Sien O teran L pe ea Mer drre mnn Sem Ov an Qm3 Cie ONS Gen ONUgse Se ma beret Come ee Dm Sandvine Soe 1 2 9 0000 eet o poc A mx 0n Ser ewe ox poo Mret I I i Proe A mna iex Loews 1 a rfet Ow Laa X nm mI ret 1 Mee o ecient Ow L I Pioc ji i nee beres pa i Few ow oon Uie Las i ptc met 8 1e 2008 rrr 3 joe oem 10 rwt 1008 m eue 7 I I Bowie ries 7 Stem 01 OWENS tomemos 53 etet os 1 i 00M iex erma 3 BOWEN TRT bos refet os boo beie 120 300 3 I0W1 w2 tomate X denied 0 0000X 6 be 162008 reer 1 STZ bomaro Ds Ardeo Sen o I Dou OrieX ed 1 TZ omes Dus ae s c I I Cite eee yw i Inse Tap bao Uee 1X mw eaer X Pet he Domen Driexo 120 306 1 tama lee XP Urim s i Won e Trot Pet Pe bios oses Soe 1 I9 Tr
62. a certain size within the database Sorting the data will simply order the data for example it will order the data in size order with small aberrations first and large aberrations last The procedure is as follows 1 In the database management screen click on the data that is to be filtered or sorted TODOS Genomic View Table View Aberrations Database Management Program Files CytoSure Analys Table View O Ideogram View O QC Trends Group data by Submission Submissions ScenDate amp Aber Subm 7 File Location ArayBa C Array 7o SamplelD Sample Phenoty 7 MACAd 7 UserName QC Metrics 12 Tue Dec 02 12 Pritest XDBUL Syndrome Plus 00 1A A C8 scan 06 14 2008 1 13 Tue Dec 02 12 X A0184 cohid Syndrome Plus 00 1A A0 CB scan 12 12 2008 0 12 Fri Dec 12 15 PAO184 cohid 0 1A AQ C8 scan D Fri Dec 12 15 PADISe cohid 00 1A A0 C8 scan 06 14 2008 1 12 Fri Dec 12 151 PAtest XDELL Syndrome Plus gaetgedg 00 1A AO C8 scan 08 14 2008 1 13 Fri Jan 02 LZ PihtestiXDEU Syndrome Plus 00 1A A0 C8 scan 06 14 2008 1 28 Wed Jan 07 1 PAtest XDELL Syndrome Plus egrgwer 1A AD CB scan 11 05 2008 1 1 Tue Jan 20 10 PAD184 cghld sample 1A AQ CB scan 11 05 2006 1 3 Tue Jan 20 10 P0184 cghid sample 00 14 A0 C8 scan waegequawe
63. add custom annotation tracks select Tools gt Options gt Annotation Options Eg Files ces Annotation Classification Report Generation Database Display Genome Normalisation Probe Filtering Custom Annotation Tracks Import new track Remove selected Edit selected Initial Colour File Genome Annotation Filters Annotation Type Field contains 3 does nat contain Create Filter Remove Selected Applv Changes and Close Figure 18 Adding custom annotation To add custom annotation prepare a txt using Microsoft Excel The file needs to have columns containing the following data Chromosome Start Stop and Number or Sample ID An image of an appropriate txt file opened in Excel is shown below 990055 Version 11 28 A Number 106 129 218 263 611 750 881 890 894 983 991 1015 1186 1389 1458 15 5 1588 1617 1634 1803 1925 1927 B Chromosome e e e KA MA EF FEF EF FP RP EP FE BF FIR Kk FE FF Ka Start Position bp 918164 175311915 3214534 145434722 219787706 190142199 105468369 145434722 941583 144149973 216532635 2186983622 145434722 144979554 246013826 108330015 215209972 120270573 20428363 18255166 144807807 69875752 Figure 19 Adding an annotation track End position bp oft Arraytype 1145847 1Mb Clone Array 187408224 Tilepath cloneset 6042798 1Mb Clone Array 146342686 1Mb Clone Array 22189050
64. aen FlestCytoS C Table View C Ideogr arn View Group data by Submission C5 Aberration Refresh Subenis siones Scan Date Z Aber Submiss F e Location Array B Array D Sample ID Sample Pheenoty MAC Ad User Name QC Metrics 06 14 2008 14 1 Tue Feb 17 15 iv MestXxDeu o Syndrome Plus 000 01 6C 0O E DouglasH 01 06 2009 16 3 Fri Feb 27 15 PA0184 cghid 00 01 6C 0O E Douglas 4 17 xy Wied May O6 1 lt 9 t OOS OX E Douckaud Abert ations Che Rat Stop See Classi CNY Gand CNY Reg Syndr Import Decipher Aberrations P Value Gary Figure 88 Highlighting the relevant datasets QC metrics will be plotted on a graph The X axis can be plotted in sequential order or by date 990055 Version 11 98 Oxford Gene Technology taw Normalised Smoothe jisplay Filtere R post RT_25226661 _no norm_2_4 txt I Invert w a Raw Normalised Sm o hed Display Filterec J JJ 2 o d v Database Management Genome Genomic view Table view Figure 89 QC metric plots 7 7 Backing up the database file To backup the database click on the Backup button see diagram A backup database file will then be created to ensure that there is a copy in case the original db xml becomes corrupted In order to position the backup file use Tools Options Files Database Backup Location If a previous version of the database is required click Restor
65. ap 9 Ow Grm 10 vereer 3 i LAE oon Ob 162008 qe x6 31 hot Tart D undae 100 Veen D Poet Pun oe r U pe N 1 ie qe o9 lade Pv I New PO Peet rer bone ries teem 1 aeneo vei D rafed 100 x Wee 6 Peet re bou iex yw 3 Je wen D Uee 100 X Neue K I Pee tm Soomro ies E Uwe ees bo rse 100 0 Meum ol I Fet rar X rani 210808 7 E pene Seo Bir irde 100 AT Yee X i Pet tm Den9w L ea 2 eo Dow bo jumdeefed 1009 Ea n Pe Sios OF 1h 008 rr umo suno Kang Lre 100 De mee CAPIO Puer re R Rnz OPiexo O X e Mem d Bis Unimed os I i i D iex LL Iw i e Dis Undead 0 I 7 WFO amp OM1e2008 Mex N emm o ae Bs ee os e 7 Yat iex c spa 95 17 2 TRN Dis rfe Ow c I Syry 00 16 2008 0 othe TR bI ON mmi 100 E Newer X Tiare Dx fux XA dE 10 Urde 1009 000 Wehen X Peers Pun kaose T Dero Y aset een D rafet 1009 am Veen HZ i bem oou Ori a 1741 rw eo Undeefed 1009 wow TON LS ae LIT E bars ion 645 reatet Wen 9 core owe BN iex ew Pas 7 15 lected 100 I Veen 6 cores LE Bonna Bele 7 L Pec ae E I aS Leet 1009 i Wontar XZ cores tome XZT Sie Mex X nec Yo 8615 tented 1009 E Veena 20 cores tome CC iex C whole eM T ean ae o ewm 7 rerna seos 1 a emm rere E gt Figure 80 Radio button to click to display the database information as an ideogram CytoSure Analysts Sof
66. are o 4 mum S M L ee ee ee DI Cw Rant 2 os 1 199790299 199971677 0 09 e y 1 195009719 195046569 0 08 Z SOO L 0 11 3 164006109 164081599 0 07 S 180396523 190063154 0 037 amp 1229504 12295524 0 0 10 81657209 1641986 0 0 10 81649597 81943864 0 27 ebay Sample Fle Assocation L i Z i e o ww wiwiNiwit amp qi eiui wie m yi elwio anny uw ani 252009210123 OTOI 501 OGT 17 7 2008 2 1 2 252009210123 LOTO 501 0GT 17 7 2008 2 1 1 252009210124 ROT 01 061 17 7 2008 2 1 2 0 lt 9 e 2 Z 172992 17299967 0 0 22 17318160 17366108 0 04 23 i24 IMTS 00 DTE zy 2182622 224098 6 07 2 Ix 2104007 WO 2219388 2525780 04 10 1644944 164290 00 3 Figure 72 Using the method of combining the datasets before analysis The software will combine the datasets in such a way that the aberrations that have been called correctly by both arrays will have a high value Those that have only been called in one dataset will have a low value The results are then outputted as shown below 990055 Version 11 85 Oxford Gene Technology CytoSure Analysis Software File View Tools Help 2008_2_1_2 txt B Invert TERI R Invert ps P2 228 weis 08 2 1 2 txt I Invert Stop Classification 13646742 D in RB E SMS G39ESNN SMEARSRRA a e a E E E 10 FEA TN E Uy Um S in 54 i 15 S l E
67. ata Edit Kd Tick to close the datset Source File post ET 252266610008 501 4 BX Design Type l Mapped Probes X Separation DLR Spread v fe p gt Aberrations Chr Start Stop Mb x 164878 2524916 2 36 15 88476955 92191463 3 71 22 129258883 130054858 0 79 Figure 5 The tabs and the radio buttons 990055 Version 11 13 Opening previously analysed files Files that have been previously analysed using CytoSure analysis software and saved can be reopened in CytoSure analysis software by using File gt Open 4 Viewing CytoSure data Once data files have been loaded the user is able to browse the data using a number of controls supplied by each component of the interface 990055 Version 11 14 Oxford Gene Technology ogt File View Tools Help E HIM Load B Genome Genomic View Table View Aberrations Database Management Population Analysis i Chromosome 3 TELE Raw Normalised Smoothed Display Filtered o E J E 4 41 Sample Information ES fes adden 9 sample ID G 1 i N 5 Experiment Sample M vs Reference M N as S ater secpa an pase Seu mane en pono podes Ev ee eNO ar TASS Eee DAN eoe die denter Od EIE Sample Details a Chromosome Section S e m Jm Phenotype Details Inv K 2 0 0Mb 66 482275Mb 132 96455133333333Mb 199 446827Mb Fi g ZU S1
68. atabase is the DGV The plot is shown in figure 16 EB S G S 00 Bap Oe E EE ELS E EO MINE CU UE IN ll NN 1 Hide Copy Number Variation Track Show Frequency Plat v E cl IN m Al uu Figure 15 To select the frequency plot right click in the annotation track and select the show frequency plot option 990055 Version 11 26 ogt Genom View Table View Aberrations Database Management Loop Analyss Chromosome 15 30 1447300 32 082614ub M 0204900 35 SE H 190 probes depiayod j rupi lt a e xm 1141 1 14 b Wis EU HEU NAMSA N ol Hi MINI Inthese areas of the genome there are many CN sin the DGV Figure 16 View of the frequency plot Chromosome 15 TM Me MIB BN 79 annotation tracks S To remove an annotation track B npphsbsos bem meii is ew s ewig right click in the relevant track S j outside a bar see diagram below E s A pop up box will appear Hide a an confirmation track Click this to 48 327 382Mb 54 901061 Mb amp 1 474741Mb amp 8 048421HMb remove the relevant track Figure 17 Hiding tracks 139 probes displayed Sl VUM AE RUE HD aT H OA AO IU H vU EN ITI IIT E E I EEUU MOSS tl m v lt carrera Trak Patient 990055 Version 11 27 Adding back annotation tracks Users can set up their own annotation tracks containing for example data obtained on BAC arrays or by Karyotyping To
69. ation 34580 Variati Thrombocytopenia absent radius syndrome 88 90 Patient Pati 2 23652424 j Unclassified 0 Wariation_6785 Variation_8329 Variation 4255 Variatio 1q21 1 recurrent microduplication possible suscepti GJ45 GIA Patient Pati 1 11696258 7 Unclassified 32 Wariation_1087 Variation 7448 Variation 2524 Variation Rati Patia 1 0 j Unclassified 32 Wariation 1392 Variation 1083 Variation 1087 variatio paat Bab 241930055 T 7 Unclassified 100 100 Variation 2524 Variation 38138 Variation 7448 Variat tier Patia 24055 Panta j Unclassified 27 Mariation 5372 Variation 4424 Variation 9012 Yariatio Patient Patia 5 78266212 Tu LM M NNNM D Variation 32724 Variation 0283 Variatie a 0 100 Variation 7501 Variation 31241 Variation O70 Variat ooro T B En 32724 Variation 7501 Variation 0710 Variatie a O A A S Qnis 4 OO ee ara S O S DRS e A DR s Windassfied dmm nas S S o 3 Unclassified 35 Wariation 36816 Variation 36815 Variation 1462 Varia 828 s desede Tp Taiga 0 a Unclassified 100 Wariation 4622 Variation 31491 Variation 2817 Variati haet o 102864776 100 Variation 2817 Variation 33103 Variation 38097 Variat 8 Unclassified 100 iaaio 4624 Variation 2817 Variation 4622 Variation ara H Unclassified 100 Wariation_2137 Variation 4633 Variation 463
70. base This tab allows the user to review and if necessary edit the database information I Population Analysis The software has the ability to analyse all the stored aberrations in the database Users can analyse the frequency of aberrations across a population J Workflow panel The workflow feature is an easy use method of using the CytoSure software It consist of a number of sequential steps which are followed by clicking the buttons on the Workflow panel 990055 Version 11 9 K Sample detail panel Sample details and QC metrics and present here in this panel L Aberration panel The aberrations called as a consequence of running the CBS or by calling manually are shown here M Display adjustment toolbar The toolbar provides a number of controls allowing the user to adjust the display to their requirements 3 Loading data files Opening feature extracted files To display CytoSure data the user must load a feature extracted file The file should contain red and green signal values for each probe in the array being analysed The software supports both Agilent and Axon GenePix format feature extracted files 990055 Version 11 10 Figure 2 Importing data files For analysis of mouse or rat files click File gt Select genome and select the relevant genome annotation The default annotation is human To import a file click File gt Import Select the feature extracted txt file you wish to import and click
71. berration are visible on the screen C Click Add Repeat for other aberrations in the chromosome It is important to include all aberrations on the same chromosome see discussion below 990055 Version 11 129 ogt Genome im Display Filtered Imu ren Pl N taw Normalised Smoothed LER Xm Sample Information MEPA suo E QJ m E 5 B Sample ID EIN 1 N i Experiment Sample M vs Reference M v 3 Sample Details i fe Chromosome Section S d gt 164 042738Mb 164 068009Mb E 164 093281Mb 164 118553Mb ba 210 E Phenotype Details E a N 3 Inv 11 8 12 X a E E m 21315 14 2 L M i N lt x G s lB N i 15 a 16 Aberrations wo E 1 L Chr Start Stop Mb e i 17 J 18 3 164067097 164093831 0 02 SI l 5 a hy Pal 19 7 20 Inv 21 22 i 10 probes displayed v Y V Variation_4361 2 BL Patient 1989 a Pes Genome l i CNVR1608 1 emove xpor Classify Click on the classify button to progress to the next stage This is the process where the user needs to classify the aberration according to whether the aberration is a CNV ie not involved in disease or a potential pathogenic aberration that requires further investigation The software has a suite of tools to help Principally the following questions need to be answered 1 Is the aberration covered by a CNV entry in the DGV If it is then it is likely to b
72. ccessed directly in the workflow using the Load button Display of files in the workflow Files that are present in the workflow mode are marked with a star T T 00019992Zsz lt Kd lt eg 3 Process 990055 Version 11 125 Process Wey On clicking the process button the data will undergo CBS processing and then the aberrations will be called using a threshold This step will be faster on a quad core computer see system requirements and should take about 3 4 minutes for processing a 2x105k Syndrome Plus array 4 Describe gt Describe During this step the user can type in the sample Id the sample description and sample phenotype Additional information can be added by clicking on the tabs If more than one dataset is loaded then the filename will be highlighted with the relevant colour and once update is pressed a second box will appear When complete click Update 990055 Version 11 126 ogt Sample Details 252009210003_OGT 17 7 2008_2 1 1 txt Sample Label O Cy3 CB Sample Gender Male Female Sample ID Sample Details Phenotype Details 5 Review Review IA The aberrations that the software has detected can then be reviewed by the user Manual aberration detection can be used to either add additional aberrations or delete them This process is described below a Locate the aberration in either the chromosome view
73. d above Once the changes have been made click Apply changes and Close It is possible to set your own classification terms Please see customisation of classification terms below 990055 Version 11 63 ogt Genome Genomic view Table View Aberrations Size Mb Scan Date Array Bar Array De Sample D LEI M 1 45075972 45083809 0 0070 08 14 2008 Syndrome PI E 08157762 109665904 0 508 08 14 2008 Syndrome Pl 147306825 147644801 Teso E RERO a 2 06 14 2008 Syndrome Pl 2 14 2008 Syndrome pL View Aberration 9 S056 08 14 2008 syndrome Pla S pas ERA es E R pues IS ess hesa her ea T 15 19988076 oDIzOI0 Res aasa T Syndrome P 15 ennes enoa pos erez Syndrome Fl 15 ensem piesni oaea Renan T Syndrome Pl 1e SoZ70998 G2862289 S91 _fosaa 200s Syndrome Pl 16 EG2H439 E3604264 389 J amp l4 2008 Syndrome Pl ED j E DME WI Wei T HHT aH m EY TA nin E TITO m N n La w m EN miu E En my INE E Wait E un GI Te TE LLLE TER N N Whole Genome 1 i lii gt Figure 54 Right clicking on the aberration in the Aberration view 990055 Version 11 64 Oxford Gene Technology ogt ETT C eH u BN Raw Normalised De Noised Smoothed Aberration Details x Ld o E Genome Aberration Details X Separation
74. data points select Tools gt Options Choose colour 1 or colour 2 and click Edit Select colour required from the palette and click Ok CytoSure Viewer PE File View Tools Help I aw Normalised De Noised Smoothed H H J o a Options erty Display Files Tools X Separation E 2 File 1 0 781 758 z B Scatter Plot Display e es E C Automatic Signal Axis Rescaling SS EET TR EE Data Point Size Ej Orientation Vertical Signal Axis E 9 Horizontal Signal Axis Tar ee Oooo peser Emm MELLE MNNEHEEESEN mee Pee 992 IT Iaa H izismmm m SIR nn 1 1E 997 Colour 1 ill doe oe 2 oe 0 Colour 2 E m A oo a IC DET d Preview mn L1 s Sample Text Sample Text E E U Sample Text Sample Text 3 584257Mb 9 467815333 Figure 31 Options for data point colours 990055 Version 11 41 Altering the data points sizes Select Tools gt Options and select the data point size required Click Ok Turning on off axis rescaling CytoSure automatically rescales the y axis depending on the ratio values in that region This can be confusing if comparing data points from different parts of the genome The rescaling can be turned off by selecting Tools gt Options and checking the relevant radio button Axis options There are several options in View that will rescale the scatter plot display either increasing or reducing the y axis or toggling up and down the a
75. e a CNV 990055 Version 11 130 oft 2 Is the aberration where a Syndrome aberration is If it is then it is likely to be pathogenic 3 Does Decipher or Ecaruca contain similar aberrations If so then the aberration could be pathogenic 4 Do any of the function of genes suggest they might be involved in disease If so then the aberration might be pathogenic 5 Does the aberration have important genes If it does then the aberration may be pathogenic 6 Have you seen the aberration before If you have with similar phenotype then the aberration might be pathogenic The annotation tracks are in key tool in answering many of the questions They are described in more detail in section 4 2 Question 1 To determine if the aberration might be a CNV then the annotation tracks are used The detailed use of the annotation tracks is described in section 4 2 The DGV track dark red contains information about whether a particular aberration is present in the normal population Therefore if there is good coverage of an aberration in this track then the aberration should be covered as a CNV Question 2 The Syndrome track dark blue track shows the position of aberrations found in common cytogenetic syndromes Clicking on the block will take the users to more information about the syndrome Question 3 Decipher and Ecaruca are on line databases containing information about cytogenetic aberrations There are Decipher and Ecaruca
76. e and a box will appear listing previously backed up database files Double click on one of the files listed to carry out the restoration 990055 Version 11 99 Oxford Gene Technology Figure 90 Backing up the database file 7 8 Exporting data in the database There is now the ability to export the data from the database to a tab delimeted ixt file Clicking on the Export button in the Database Management tab will export the data The txt file can be opened using for example Microsoft Excel 990055 Version 11 100 8 Population analysis 8 1 Grouping data and plotting aberration frequencies The Population Analysis tab provides calculation and graphing of aberration frequencies from groups derived from your data For example it can enable you to query the database for the following data a Which aberrations are present in my population with a different frequency in autistic samples compared to non autistic samples b Does this frequency vary in one ethnic group compared to an alternative ethnic group Question 1 For population analysis it is important to type in the phenotype consistently in the phenotype box o0 this can be done by either Import the txt or gpr files into the software Using File Properties Sample details Editing the Phenotype Details field in the database Figure 91 shows a view of a database with a number of samples containing the word autism in their Phenotype Details field Note that
77. e extract with CytoSure algorithm ogt Chromosome Section Normalise 9 Would you like to normalise the data we Click Yes to normalise the data To skip normalisation click No Raw Normalised Smoothed E o Sample InFormation Display Filtered Figure 4 The normalisation query box Loading in multiple files 990055 Version 11 Sample ID Experiment Sample M vs Reference F Sample Details Phenotype Details Aberrations Chr Start Stop Mb Remove Export Multiple files can be loaded in by sequentially using File gt Import The different arrays results will be displayed in different colours Note that data from more than 2 arrays may cause the computer to become slow depending on the memory of the computer used When there a multiple datasets loaded a series of tabs will appear on the right hand side of the screen Clicking on the tabs will toggle between the sample details of each dataset Associated with each tab are some tick boxes These are shown in the figure below TOM C Display Raw Normalised Smoothed Filtered 3 Q Sample Information Poste REL N 4 Tab Click here to display sample details Experiment Sample M Sample Details qdfwerqwe N O Inv Ticked if dataset displayed Phenotype Details v wefertert n N a N S Tick to invert the d
78. ee Ss SS EI ELE PLUME LE I TEE NN o LE IL LE OEIL T 9 BII uii TUE INV NEE NN 28 MN EI NEM NBI simi N N lt 3 Whole f Genome File 3 0 019338764032594456 X Separation File 1 0 03802084799421313 File 2 0 05282440099787219 1 2 3 Chr Start 108598877 70341301 7181236 42114906 81644844 18677217 19988158 16 31974463 22 15732051 SIT 2128264 X 142618255 Stop 108862771 70345683 7790006 42135021 81943864 19465583 19992424 33830849 15749206 2413341 0 142889011 0 N on oe 10 10 15 15 Figure 64 Running the loop analysis A dialog box will appear that will allow the user to input the sample details and with which dye the samples were labelled If some of the sample details were inputted during file input then some of these details will be automatically generated Under analysis method click Use CBS calls see figure 65 990055 Version 11 79 ogt CytoSure Analysis Software 13649617 Loss in RB P Be Lon i EN Hao Lon n SA UT Gan in EB 5957 Lows e EN 07736 Gan in EN 53645 Loss n SA COOL Loss in SA 2651710 in SA 03724 Loss in SA 19739 Lows n SA 57138 Loss in SA bat 7 how i SA Smp Fe Aiboa E2595 Loss 6 5A 252009010123 SLOTO1 501 0GT 17 7 2008 2 1 2 bt BN nr ys on in SA 25301 Loss in S 252009210123
79. emale DNA when the Chromosome X probes are being used as a control Other array metrics such as mean signal intensity are available by clicking File gt Properties Exporting data and images Data from CytoSure can be exported by using File gt Export A choice is then offered between an image and a text file The text file contains the normalised data File defaults 990055 Version 11 134 CytoSure uses default settings for importing and saving files These default locations can be altered by clicking Tools gt Options and then clicking on the Files tab Click on and navigate to the folder required Options x Display Genome Normalisation Probe Filtering Aberration Detection Annotation Classification Report Generation Database Files Protocols Automatically Save Automatically save cgh data File during protocol Save in the same location as the raw data File O Save in the default save location Mame using Raw data filename Sample ID Barcode Default Save Location C Program Files CytoSure Analysis Software Default Import Location C Program Files CytoSure Analysis Software Default Open Location C Program Files CytoSure Analysis Software Default Export Location oA H C Program Files CytoSure Analysis Software Apply Changes and Close 990055 Version 11 135 oft Figure 83 Setting the file defaults 13 Contact details Oxford Gene Technolo
80. ey et a Science 297 1003 1007 These regions are typically surrounded by segmental duplications Copy number variations V dark red Source Toronto DGV database This extensive database shows the position of human CNVs There may be errors in the Toronto database so these regions should only be regarded as an indication that there is a benign CNV Clicking on these bars will take the user to the relevant DGV entry Confirmation bar C black This displays the position of various publically available FISH BAC and MLPA probes that can be used for confirmation Database track A blue This shows the positions of aberrations recorded by previous experiments which have been saved in the database Double clicking on the bar will display an image and annotation of the aberration that has been saved to the database figure 10 990055 Version 11 22 CytoSure XDB Aberration Details File View Tools Help Aberration Details Genome I p Um Can 248926 14857754 Size Mb 14 608 Scan Date Sample Details Phenotype Details Gain Loss Loss Figure 12 Aberration details of an aberration that has been saved to the database Decipher D red green Position of patient deletions Red and duplications Green from the Decipher database https decipher sanger ac uk Click on the bar to access the relevant Decipher page For up to date information please access the Decipher web site directly
81. ff the display of the data 990055 Version 11 18 The close data button will remove the data from the CytoSure software following a warning At the bottom of the chromosome section view C is the annotation Further details are supplied in section 4 2 Finally the cytogenetic location is displayed with the grey lines Whole Genome view Clicking on the HESS will display the whole genome view Clicking on the EEE button will then display the whole genome where the probes are plotted in sequential order rather than their genomic position In this view it is often easier to locate aberrations 990055 Version 11 19 Oxford Gene Technology ost CytoSure Interpret File View Tools Help Genome Genomic View Table View Aberrations Database Management Population Analysis Whole Genome L HAS T OMNE UR Display Raw Normalised Smoothed Ha Filtered s Experiment Sample M vs Reference M J Sample Details 5054 0 10109 3333333533334 151640 L 800019992257 dD rY P Figure 9 Whole genome equally spaced view 4 2 Annotation The chromosome section view C also provides the user with a number of references to regions of interest within the selected section References are grouped into 990055 Version 11 20 Ae Lu ILI T LL Al H T 1 0 bes I Is L E UUL I h E g R n sue i TT I U a gt LM LU ELLE NI
82. graphic of the chosen chromosome and a scatter plot of og base 2 of the red green mean signal ratio loge R G for each probe against its chromosomal genomic base position If the whole genome is selected the panel indicates the chromosome boundaries in place of the chromosome graphic Note that if the invert button is ticked then the data will be displayed as loge G R See section 4 1 for more details on inverting the data C Chromosome section view This panel plots a user selected subset of the data available in the chromosome overview in the same manner loge R G against chromosomal genomic base position When data are loaded this panel also supplies the user with syndrome gene and exon references with which plotted probes can be correlated as an aid to analysis D Genomic view The Genomic view displays the graph of the log ratio versus genomic position for the probes There are also tracks showing annotation E Table view This panel displays detailed information about the probes that are visible in the chromosome section view This includes the log ratio red and green signal See section 4 9 G Aberration tab This panel displays details information about the aberration selected either by the user manually or using the automated aberration detection see section 5 The aberrations can be annotated and edited section 5 3 H Database management tab The software has the ability to store the aberration results detected in a data
83. gy Begbroke Science Park Sandy Lane Yarnton Oxford OX5 1PF UK Oxford Gene Technology Operations Ltd Registered in England No 03845432 Begbroke Science Park Sandy Lane Yarnton Oxford OX5 1PF UK T 44 0 1865 856826 F 44 0 1865 848684 E products ogt co uk W www ogt co uk Technical support E support ogt co uk 14 Legal Information Trademarks OGT CytoSure 990055 Version 11 136
84. he chromosome as the baseline This could be affected by large aberrant regions in the chromosome Zero will set the baseline at zero whatever the data is Chromosome Mean log probes Uses the mean value of all the probes as the baseline Chromosome Median log probes Uses the median value of all probes as the baseline Annotation settings In this section the user can alter the information displayed in the annotation tracks 990055 Version 11 116 Options EJ Classification Report Generation Database Files Display Genome Normalisation Probe Filtering CBS Annotation Custom Annotation Tracks Import new track Remove selected Edit selected Initial Colour File Genome Annotation Filters Annotation Type Field v contains 3 does nat contain Create Filter Remove Selected Filter Applv Changes and Close Custom annotation track The user can add a custom annotation track The user will have to create a txt file with a header containing Chr Start Stop ID Then click Import New track and navigate to the relevant file More details about how to set up custom annotation tracks are described in section 4 2 Annotation Filters The annotation can be filtered to remove certain entries For example from the CNV DGV annotation the user may wish to remove CNVs that are inversions Detailed information about annotation track filtering is given in section 4 2 990055 Version
85. ification that can be used in the aberration table are shown below 990055 Version 11 ogt 68 Genomic View Table View Aberrations Database Management Population Analysis Table View Ideogram View Chr Start Stop Cytogene Size Mb Classification CNY GainlLoss DB CNW DB Gain L CNY Regions 36 CNY seeni 61 3 0 Yo Variation_33 E 2994 y m View Aberration Edit Aberration Details Classify gt 9 pcny de novo parental origin of rearrangement undefined de novo arising on maternal chromosome de novo arising on paternal chromosome Familial inherited From normal mother and Father Familial pat inherited From normal Father Familial mat inherited from normal mother Familial mat inherited from mother with similar phenotype to child CNV seen in normal individuals CNV parents not analysed CNV parents analysed seen in parents CNV parents analysed not seen in parents Familial parental origin unknown Waiting on Parents Unclassified Figure 59 new classification added 5 5 4 Saving the file To save the Circular Binary Segmentation the aberrations and the annotation use File gt Save Saving of multiple datasets could be limited by the memory capacity of the computer To save 3 datasets it is recommended that the computer has a memory capacity of 3 to 4 GB 5 5 5 Saving to the database To save the annotated aberrations and images to the database click on Save Abe
86. ifies data analysis and makes the routine analysis of aCGH data faster and more straightforward There are two stages in the process Firstly protocol set up and secondly routine data analysis In the first instance it is necessary to set up an analysis protocol This task is best suited to more experienced users who can define and select the most appropriate settings for data analysis within their particular laboratory The appropriate settings can then be saved Routine users can then load the protocol and analyse the data using the previously defined settings It is possible to alter protocol settings within Workflow mode however the software will not report that a protocol has been used in the reporting The routine user will also be guided through the workflow outlined in the diagram above The setting up of the software in Workflow mode is covered in detail in Section 10 of the user manual The use of the software in Workflow mode by a routine user is covered in Section 11 990055 Version 11 6 1 Computer requirements software installation and update It is recommended that CytoSure Interpret software is installed on a computer with Windows XP or Vista For proper display do not use Windows classic view For viewing and saving multiple datasets it is recommended that a computer with a Memory of 3 4GB is used To ensure fast running of the automated aberration detection it is recommended that the computer used has a quad core If a quad
87. ion Aberration 14 Figure 35 2D chromosomal ideogram 4 9 Table View tab 17 To obtain additional information on displayed probes d use table view Navigate in Chromosome section view to display probes desired Click on Table tab E on figure 1 This reveals relevant probe information 990055 Version 11 44 oft Genome Table View Aberrations IT mal M Probe Mame Chr Start Sequence Red Signal Green Signal Log Ratio Normalize Cad Oll 1 37536949 37537008A 3381 0 3645 0 0 10846879 0 0946401 4 GHM ia 37613804 3USIS3A 4258 0 4872 0 0 19433818 0 1631380 GH ia 3 S10896 a7s1095sla 4727 0 S169 0 0 12896083 0 0901075 Gh M ia 3 268857 37268818 S290 5668 0 0 10356327 0 0736654 Gh 4 3HiZ93 3729S7SA H TT S836 0 0 10865149 0 0753198 GH DIT d 36734582 36734641 E 3456 00 11419066 0 1026996 GH iT ia 3639090 3639149 aozo 3023 0 D 2I2I9810 D 21966541 GH DIT 36282999 36283 98A HE T 3791 0 0 32612081 0 3128943 GH DIT ia 36240988 36240097 3452 0 2995 0 D 20487646 D 20851583 GH DIT aa 36114103 ass 485z0 4584 0 D D8345544 D LIS84347 iil ficere 4 seoseuo sensisse H 3459 0 3931 0 0 18454135 0 1703369 IS a r cesmoma 28460 2641 0 0 10785136 D 11080640 CGH DIT 4 aza 359230728 2672 0 2368 0 0174250
88. ion 11 59 ogt View SEE Help Identify Aberrations Raw Normalised De Moised Smoothed De noise 451001 no norm_1_i t lt t lg Invert gt E 6 a E a Smooth Export Aberration Report amp Separation Probe Selection Tool i File 1 0 034708590955975116 2 Mormalise on current subset Merge Displayed Aberrations P San 13 882635Mb 16 823584Mb 13 804534Mb 22 715484Mb Chromosome Section ES iat Wn m m y i HEREIN HEL am M ka Co all L ba I I I 1563 probes displaved Figure 51 Merging the aberrations To undo merging place the mouse over the shaded region and right click Select dissolve 5 5 2 The Aberration tab Editing and annotating the aberration list Altering the sample details after File Import The sample details annotation can be altered after the File has been imported using File gt Properties see figure 52 or using the Edit button in the Sample details panel If using File gt properties select Click here to edit sample details button to change the sample annotation Click on the genome web view tab to refresh the aberration list screen if required 990055 Version 11 60 ogt Genomic View Table View Aberrations Chromosome 1 T TA Properties h Data Set 1i Senaration Ef 0 015195583128267849 z L2 l iubens die E I Hd Data Set 1 start Stop Mb x
89. is backed up regularly f this is done then the database needs to be linked to the db xml file as follows 1 Go to the database modification page by clicking on the database modification tab EN CytoSure Analysis Software l S Raw Normalised Smoothed SPv2 Agilent txt E Invert P o Genomic View Table View Aberrations Database Management Chromosome 1 11 an EE STI NJ LIT pe E DE S 11 LP L Figure 76 Opening database modification page 2 Click on the File address at the top left hand side of the screen figure 77 990055 Version 11 87 Oxford Gene Technology CytoSure Analysis Software l Genomic View C Program Files Cyt Group data by O Submission It Aes MES iei 2 Reis MIMESS egower 02 Dec 200 so eer e mu m me e 02 Jan 200 10650198 0 16 4 Figure 76 Mapping the db xml file location 3 Navigate to position of the moved db xml file and click open 990055 Version 11 88 ogt 7 2 Viewing data in the database Click on the database management tab to access information stored in the database CytoSure Analysis Software M af File View Tools Help m MEME 2 Raw Normalised Smoothed Display Filtered Genome Genomic View Table View Aberrations Database Management C Program Files Cy Group data by Submission Aberration Submissions Date Sample ID amp Abe
90. llows Unknown de novo parental origin of rearrangement undefined de novo arising on maternal chromosome de novo arising on paternal chromosome familial pat inherited from normal father familial mat inherited from normal mother familial mat inherited from mother with similar phenotype to child CNV seen in normal individuals CNV parents not analysed CNV parents analysed seen in parents CNV parents analysed not seen in parents Familial parental origin unknown 990055 Version 11 62 familial inherited from normal mother and father Unclassified These terms can also be customised by the user Please see section on customisation of classification terms in section 5 5 3 On classification of the aberration the row will change colour according to the classification assigned as follows e Pink CNV e Grey unclassified e Purple potential pathogenic aberration Genomic View Table View Aberrations Database Management C Program Fil Table View Ideogram C7 QC Trends Group databy Submission Aberration Restore Aberrations i E Submission Chr Start Stop Cytogene Size J Classi CNY 9 s Gain v DB CNY 95 DB GainjL CNY Regi a3 i4 17 Feb 200 1 45075972 45084438 0 0080 Unclassified 0 0 a 3 27 Feb 200 14 105481523 106329869 0 848 lUnclassified
91. lly examine TeplI gale ccncscxceussersncgneunspeavechsdudccessaseneiecimesaneidens 60 6 5 Rerunning the loop analvaig ee e e e ee e teres 62 6 6 Saving loop aberrations to the database cccc cece cece ee eee senate eee eens 62 6 7 Alternative method The combination methods Les 63 6 8 Saving the loop aberrations to the database combination method 65 Database Manadement cc css s sss ecce cce e e e e ee eee eee eee 66 7 1 Positioning the database file in a location of the user s choice 66 7 2 Viewing the data within the database esses 68 13 Deleting aberrations in the internal database Leeeuuuesus 72 7 4 Editing aberrations in the internal database cccceeseeeseeeeeeeeneens 74 239 Sorting or filtration of data in the internal database 76 7 6 Display na me Tie M T m oU UU T DT FEN Backing up the database Tie ecce ecce 78 7 8 Exporting the data from the database file eueeuueeeuuueee 79 Population ANALYSIS c ususeina e c c eee e e e c c Ru ee eee 79 8 1 Grouping data and plotting aberration frequency ssueusu 79 8 2 COMDNNA ON RETE ETT 83 8 3 Ra GS AEA T I LT ET 85 8 4 Exporting aberration frequency results and statistics issse 86 Setting up a protocol in Workflow mode
92. ltering Aberration Detection Annotation Automatically Process Automatically process using Circular Binary Segmentation during protocol Aberration Calling Options 5 yin Sie Mb ee Threshold Method Threshold Factor Chromosome 4verage Method Misc Pracess immediately after loading data skip visualisation Number of processors used during Cricular Binary Segmentation 990055 Version 11 50 ogt Figure 42 View of option settings for calling the aberrant segments Minimum Probe Count A value of 4 probes is suggested as the minimum number of probes in a segment that are required to make a call Threshold method and threshold factor This is the value that the algorithm uses to set the threshold value User definable Type the value of the logs ratio into the threshold factor box to set the threshold value that will be used to make a call Typically a logs ratio of 0 5 to 0 85 is used Standard deviation the software calculates the standard deviation of the ratio the threshold value will be the threshold factor x standard deviation of the ratios The preferred factor is 3 or 4 Deviation Log Ratio DLR the software calculates the DLR Spread of the ratio the threshold value will be the threshold factor x DLR of the ratios This typically is a factor of 3 or 4 X separation The software uses the value from the X chromosome probes This ass
93. n 11 33 oft Annotation Filters Annotation Type Field Copy Number variation v contains does not contain Create Filter Remove Selected Apply Changes and Close Figure 26 This will remove all CNVs from the annotation that have been classified as inversions Finally select Create Filter and Apply Changes and Close 4 3 Searching for a gene or probe or syndrome e A probe by clicking View gt Find Probe typing the name of the probe in the dialog that appears and clicking Ok e A gene or syndrome by clicking View gt Find Annotation typing the name of the reference in the dialog that appears and clicking Ok The text needs to match the Ensembl annotation or the syndrome annotation used in the software If not found try a synonym e A position in the genome by clicking View gt Go to position bp typing the number in the dialog box and clicking Ok 990055 Version 11 34 4 4 Data display the radio buttons Raw Normalised Smoothed Display Filtered E E d There are 4 radio buttons on the Display adjustment toolbar D This allows the user to toggle between different views of the data e Haw button shows the data un normalised e Normalised button shows the data after normalization e Smoothed button see section 4 6 e Filtered If checked the filtered probes are displayed If unchecked all data points are shown however the filtered probes
94. ndrome Plu Latvian D0 1A A0 C8 scan CHROMOSOM 08 14 2008 1 20 Thu Mar 26 11 P itest XDBIL Syndrome Plu Latvian D0 14 A0 C8 scan CHROMOSOM iz Tue Dec 02 12 P test XDB L Syndrome Plu autism 00 14 A0 C8 scan CHROMOSOM 08 14 2008 1 28 Wed Jan 07 1 P test XDBIL Syndrome Plu autism 00 14 40 C8 scan CHROMOSOM 11 05 2008 1 n Tue Jan 20 10 P 10184 cghid autism I00 1A A0 C8 scan CHROMOSOM 11 05 2008 1 3 Tue Jan 20 10 P 10184 cghid autism D0 1A AO0 CS8 scan CHROMOSOM 11 03 2008 1 3 Thu Mar 26 10 S YsoftwarelC Syndrome Plu autism D0 1A A0 C8 scan CHROMOSOM 11 03 2008 1 0 Thu Mar 26 10 S softwarelC Syndrome Plu autism D0 1A A0 C8 scan CHROMOSOM 08 14 2008 1 13 Tue Dec 02 12 X 10184 cghid Syndrome Plu 00 14 A0 C8 scan CHROMOSOM Aberrations Chr v Start Stop Cytogene Size v Classif CNY s Gain L vv DB CNY DB Gain L CNY Regions Syndr v Impor All Ge voo Decipher Aberrations P Yalue v 1 145375581 145682839 0 307 Unclassified 5 Variation 34 1921 1 recu Patient Pati 0 95971019 ils 18815719 19432994 i Unclassified 37 9 43598672 43599699 Unclassified 100 15 19988076 20012010 Unclassified 10095 9 509607 784666 Unclassified 10095 7 15 21171815 21190973 Unclassified 10095 Prader Willi 1 45075972 45083809 Unclassified 0 15 911
95. nually using automated detection or a combination of both click on the Export button This can be done either in the Genomic view or the Aberration view Export Aberration list This will generate a report 2 The report can be generated as an html file pdf file or a txt file with a separate folder for the images 3 Open the report file With the html file can be opened directly in Microsoft MS Word Alternatively Select all Copy and Paste to a new Microsoft Word document 990055 Version 11 70 4 Edit in MS Word if required to add text choose aberrations etc 5 The report includes the following information Image of the aberration with flanking probes Location of the aberration P value as discussed in section 5 8 below The number of probes in the aberration If the aberration is in a Syndromic region and OMIM link If the aberration is in the CNV database DGV database in Toronto If the aberration is in a Recombination Hotspot region as defined by Bailey et al Genes within the aberration with Ensembl link The genes located within the aberration Important genes The program searches the gene against a list of known human disease genes and any found will be highlighted as an important gene opace to add notes Customising the report The content of the report can be changed according to the user requirements To do this select Tools Options Report generation 990055 Version 11 71 9900
96. ocation axis This is actioned by using the corresponding arrow keys or the mouse wheel with the Alt key pressed The user can also alter the size of the signal ratio axis by pressing the Shift key whilst scrolling with the mouse wheel or pressing the up or down arrow keys Hovering over a reference will display the information for that reference and clicking on the reference will open a browser window and navigate to a relevant web page if the user s internet connection allows The display adjustment toolbar M provides the following controls 990055 Version 11 17 Zoom in Zoom out Raw Normalised Toggle between displaying the raw calculated normalised data or denoised data Select the calculated smoothed data Select a particular dataset when at least one has been loaded The invert tick box will flip the relevant dataset The standard default unchecked displays the data with the y axis as log2 red green With the box checked the particular dataset will be displayed with a y axis value of log2 green red Data visible C C Invert data Inv xX Close data The Tab at the right hand of the screen contains various buttons The invert tick box will flip the relevant dataset The standard default unchecked displays the data with the y axis as log2 red green With the box checked the particular dataset will be displayed with a y axis value of log2 green red The Data visible tick box will turn on and o
97. of the profile which have a similar copy number These are known as segments Venkatraman ES and Olshen AB 2007 Bioinformatics 23 6 p657 663 The relevant segments then need to be called as aberrant The call is dependent on how many probes are in the segment and the logs ratio value of the segment known as the threshold value The method to run automated detection and aberration calling is as follows 1 Click on Tools Identify Aberrations 2 Adialog box displays the option to either run the CBS on filtered data see section 4 5 or on raw data 990055 Version 11 48 Genomic View Table View Aberrations Database Management Chromosome 1 Chromosome Section 0 0Mb 2416 247 249719Mb Should the CBS Algorithm be carried out on Filtered data Click Yes to use filtered data Click No to run the algorithm on the Full dataset Figure 41 Running the CBS on either filtered or raw data 2 The CBS algorithm runs with the default settings To change the settings click Tools gt Options gt Aberration Calling Options 3 The CBS option box displays option settings for calling of the segments obtained by CBS 990055 Version 11 49 SM Options H Classification Report Generation Database Files Protocols Display Genome Normalisation Probe Fi
98. oft CytoSu cy Interpret software User guide version 11 This protocol booklet and its contents are Oxford Gene Technology Operations Limited 2008 All rights reserved Reproduction of all or any substantial part of its contents in any form is prohibited except that individual users may print or save portions of the protocol for their own personal use This licence does not permit users to incorporate the material or any substantial part of it in any other work or publication whether in hard copy or electronic or any other form In particular but without limitation no substantial part of the protocol booklet may be distributed or copied for any commercial purpose Contents 1 Computer requirements software installation and updates 7 2 Running CytoSure analysis software eee eee enn 7 3 Loading data Hle Succ iioeas cessent oes ares seottanaeresdeotsuceareresatdes EEES 10 4 Viewing CytoSure data 14 4 1 PAO NENNEN ses EE ETT 15 4 2 PISO Loren DRE E AE AEAEE A PERIURUEMEREDUDUF N PEDI RIS 20 4 2 1Adding custom annotation tracks 4 2 2 Filtering the annotation 4 3 Searching for a gene or probe or syndrome 34 4 4 Data display the radio DUN ONS sensi santet e 93d Nets patr dadas tat ht Rr es 35 4 5 SI ON or 1 Gaid NITIDE TT 35 4 6 Normalisation and RN PILAR Ie RET E TO TE D o D 37 4 7 DUBIE fe ene ols i E ne iari vd bet UAI TEN Edo IAE T
99. on autistic samples The screen is shown below 990055 Version 11 102 ogt Select either All aberrations only gains or only losses TR br rr eee Ape gars eeaeee EEE tem CMC C ELEM E EN G G GE Resolution Groups a l Selected region Plot of aberration table frequency within the database a Venn diagram Figure 92 View of population analysis page This shows all the data in the database plotted as aberration frequency in percentage versus genomic location The data can be visualised as in the genomic view so the chromosome buttons can be used to navigate through the chromosomes and the annotation tracks are functional so the position of genes can be identified The resolution can be altered by increasing or reducing the pixels per bin In this first example looking at the aberration frequency in autistic samples compared to non autistic samples the following procedure is used The first step is to group the data into autistic samples and non autistic samples 1 Click on Add New in the Groups section Filter type m x A 1 Sample sSubmission or Aberration Filter _Sample Submission 2 In this case the objective is to separate out the samples which have been labelled with autism in the Phenotype Details field from the remaining samples 990055 Version 11 103 ogt oelect Sample Submission select Phenotype Details from the drop down box type autism in the Con
100. otation Classification Report Generation Database E Files Protocols Automatically Save Automatically save cgh data File during protocol Save in the same location as the raw data File O Save in the default save location Mame using LS Raw data filename C 7 Sample ID 2 Barcode Default Save Location C Program Files CytoSure Analysis Software Default Import Location C Program Files CytoSure Analysis Software Default Open Location C Program Files CyboSure Analysis Software Default Export Location C Program Files CytoSure Analysis Software Apply Changes and Close HE H Automatically save If ticked the software will automatically save a cgh file after the reporting There are also options here to set the location where the cgh file will be saved Name using 990055 Version 11 121 There is a choice of naming the file as either the same file name as the txt or GPR file that has been used Alternative naming is by the sample ID or by the barcode of the slide The default file locations This specifies the folders where the files to be loaded into the software or alternatively where the files exported from the software should be located Protocol settings Options Display Genome Normalisation Probe Filtering CBS Classification Report Generation Database Current protocol Mane Create new protocol From current settings
101. our 1 l Colour 2 EN Colour 3 Apply Changes and Close 990055 Version 11 110 Automatic Signal Axis Rescaling This setting will toggle on or off the y axis rescaling on the graph in the genomic view Data Point Size Altering the setting changes the size of the datapoint in the genomic view Orientation This user can select the orientation of the graph It can either be in horizontal or vertical view see section 4 8 Karyotype Band Rendering This sets the shading in the chromosome diagram on the ideogram Colour Scheme Allows the user to set the colour of the datapoints Genome Setting Options D Classification Report Generation Database Files Protocols Display f Genome Normalisation Probe Filtering CBS Annotation Genome Type LS Human O Rat Mouse Apply Changes and Close 990055 Version 11 111 The user can set the type of annotation used Normalisation setting Options Classification ion Database Files Protocals Display Genome Normalisation Probe Filtering CBS Annotation Automatically Marmalise Automatically narmalise during protocal Normalisation Method 5 Global Normalisation 3 Local Normalisation Global Normalisation Settings Normalisation Window Size 5 000 Local Normalisation Regions chromosome s sO a pn Add Region Remove Regionis Chromosome
102. owing e Select a folder to store the processed data e Add the file to the Files to be processed area If the files are in GenePix format then in the file type drop down menu All files needs to be selected to display the GPR files e Select number of threads to be used The more threads used the faster the processing however alternative processes on the computer will be slower A quad computer using three threads should take approximately 5 minutes to process data from a Syndrome Plus 2x105k array e Select whether the data should be normalised or not e Select whether the data used should be filtered or not 990055 Version 11 53 ogt e Click Process 3 The processing will begin and the data saved to the appropriate folder Batch Processing QutEputE Location Prokacal Files to be processed Date Processed Status Add Files Remove Files Threads 6 Run on filtered data Normalise data Load into workflow Process Figure 45 CBS batch processing box 990055 Version 11 54 Oxford Gene Technology 5 3 Manual aberration detection Manual aberration detection occurs when CBS processing is not selected 1 Locate the aberration in either the chromosome view or whole genome view E JS Load B E Genome Genomic View Table View Aberrations Database Management Population Analysis Chromosome 3 Display Normalised Smoothed Filtered o
103. pply Changes and Close Figure 23 Adding an annotation track Filtering the Annotation The ability to filter an annotation track has been included in this software release This functionality now enables users to select annotation data for removal For example in the DGV CNV track the user might wish to remove CNVs that are inversions Alternatively the user may wish to avoid having BAC data in the track To filter an annotation track select Tools gt Options gt Annotation 990055 Version 11 32 ogt Options Eg Classification Report Generation Database Files Protocols Display Genome Normalisation Probe Filtering CBS b nnokation Custom Annotation Tracks Import new track Remove selected Edit selected Colour File Annotation Filters Annotation Type Field contains does nat contain Create Filter Remove Selected Filter Applv Changes and Close Figure 25 Select annotation track Select the Annotation Type to be filtered In the example shown in the figure below the Copy Number Variation Track has been selected from the Annotation Type drop down menu Then using the drop down menu click the field or column where the data is going to be filtered In this example in order to remove CNVs that are Inversions select the field Type and then select the radio button does not contain and type inversion in the search term box 990055 Versio
104. rrations User File Mame 14 Jan 2009 EW 360379 12 pbrady admin Agilent an2009 16 282807 Sfpbradyo Acre Tan 2008 maos v0 386506 aperea 2 79H 14 Jan 2009 JK 390331 2 pbradyn x tAdmintAgilent 14 Jan 2009 SU 365265 183pbradyn x tAdmintAgilent 14 Jan 2009 YH 373702 i6pbradyn x tAdmintAgilent 14 Jan 2009 AC 333961 i5pbradyn x tAdmintAgilent 4 Aberrations Start Stop Size Mb Classifica CMV Gainf Loss 19447358 19494420 0 047 Unclassified 100 49590150 43599699 0 0 iUnclassified 100 1135376 1136269 o0 Undassfied 100 Whole gt Genome 4 mE 4 Figure 77 The database management page The data that has been saved can be viewed by submission date when the data was saved to the database or by aberration see figure below If viewed by aberration the data can be sorted for example by chromosome by clicking on the appropriate column header 990055 Version 11 89 Oxford Gene Technology ogt CytoSure Analysis Software File view Tools Help Display Filtered Smoothed s P Raw Normalisec B 008 1539 A txt B Invert JA D Le Genome Genomic View Table View Aberrations Database Management Sie ee tie eee fe meee CLE T J N LP W ULT IE N egrgwer sample sample e aan L N LEITE egrgwer O2
105. rrations to Database at the bottom of the screen see figure 60 below 990055 Version 11 69 oft CytoSure XDB Seles File View Tools Help Raw Normalised De Moised Smoothed 252009210003 COGT 7 2008 2 1 1 bxb Bg Invert a ad Stee Genomic View Table View Aberrations H 1 N Size Mb Scan Date Array Bar Array De Sample D 71630600 4 646 08 14 2008 Syndrome Pl 4 a 02993187 04929149 fis amp 12008 Syndrome Pl 8 porze sa eor eres Syndrome P i4 9745808 e9ez08s0 QNS e1ez008 Syndrome Pl a fosass16 1091593 0 252 osie2008 Syndrome Pl x p4sisisz Peooesia Lz5d 914200 Syndrome Pl LLELI T wfe UT 19311 A HR 1 IN wg EN j E ud JMISLET BILIET afi RJ um IBI E BRS SGT a Rp Te NN W maj Gh 818 N N TD Hn 5m E E P NI ra lt ES a A in i hole 4 gt Genome Save Aberrations to Database Export Aberratian List in Decipher F Export Aberration Report Figure 60 Saving aberration annotation and images to the Database Saving data to the database has two effects 1 The aberrations will be displayed in the aberration track within the Genomic View tab 2 Anew entry will appear in the Database Management tab 5 6 Exporting aberration results for printing 1 Following generation of an aberration list either ma
106. s follows Array 1 Sample 1 versus Sample 2 Array 2 Sample 2 versus Sample 3 Array 3 Sample 3 versus Sample 1 CytoSure software supports this experimental design There are two different methods available to analyse the loop design The first method uses the CBS calls of all 3 datasets In this method each array is analysed and the aberrations are called The aberrations are then combined The workflow is as follows Step 1 Import the 3 arrays and run the CBS Step 2 Check the aberration called for each sample otep 3 Run the loop analysis otep 4 Manually examine the replication Step 5 Rerun the loop analysis This method is described in figure 6 1 6 2 6 3 and 6 4 The second method combines the 2 relevant replicate datasets before calling the aberrations The workflow is as follows Step 1 Import the 3 arrays otep 2 Run the loop analysis and combine the replicates otep 3 Examine the called aberrations This combination method is described in Section 6 5 990055 Version 11 76 6 1 Import the 3 arrays and run the CBS The 3 feature extracted files txt from Agilent scanner gpr from Axon scanner are imported into CytoSure software using File gt Import see section 3 The Cy5 sample details are inputted The Cy3 samples details do not need to be inputted at this stage figure 49 CytoSure Analysis Software Figure 62 Import of the array data Once the 3 datasets are imported into
107. section 6 3 This will refresh the loop analysis table 6 6 Saving aberrations to the database To save the aberrations in the database click on the aberration tab to view all the aberrations The aberration view will list the aberrations from the 3 arrays both the original CBS calls and also the loop analysis results Therefore if the user wants to save in the database only the aberrations from the loop analysis click on the drop down menu see figure below Select Automatic CBS and then Remove all aberrations of the selected type The aberrations can then be stored in the database as described in section 5 4 990055 Version 11 83 oft able View Aberrations Database Management Loop Analysis Table view Ideogram View Automatic CB5 Remove all aberrations of the selected type Mb Classification CNY Gain Loss CNY Regions Syndrome Faltas ip s Important Decipher Aber P Walue 10 Unclassified 100 Variation 2294 1p36 micradelLOOP Analysis 0 00759609 3 Unclassified 93 Wariation_30374 Variation D380 Variation 8321 Variati 1p36 deletion f S Wahai 100 Waren A Pe Pdi NB Y Wahai T Waren 2524 Variation CJ Varaton 3MIB A PeePdi TERI Unclassified 10 iaaio 8330 Variation 4254 Variation 30427 Variati 1g21 1 recurrent microduplication possible suscepti GJA GIAS Patient Pati 319682925 i Unclassified 100 58 Variation 1326 Variation 6784 Vari
108. somal aberrations For more details on the particular aberrations please visit http agserver01 azn nl 8080 ecaruca ecaruca jsp Unfortunately there is no direct link to Ecaruca entries By default this track is turned off To turn on the track see the section on adding back annotation tracks below Having many tracks open can slow the software Some of the annotation tracks can be filtered to only display certain information Please see section below on annotation filtering Expanding the annotation bars To view the full annotation click on the button shown in the diagram below mill L M Omer Figure 11 Method to enlarge IU Vll Il the annotation bars Click Full CNV annotation expanded 990055 Version 11 24 Chromosome Section 35 S58366Mb 30 144 738Mb 32 082614Mb 34 02048Mb Patient Patient Figure 13 CNV annotation displayed The annotation is displayed figure 13 and in order to view all the annotation the slider bar can be used to scroll through the annotation figure 14 pum oM ee BRL as TlacaCcon Figure 14 View of the slider enables scrolling through the annotation 990055 Version 11 25 To summarise the data a frequency plot can be plotted Right click in the relevant annotation track and select the option show frequency plot figure 15 The frequency plot is a graph where the y axis is the frequency of entries in the database for CNVs the d
109. sors used during Cricular Binary Segmentation The CBS settings can be altered here Minimum Probe Count A value of 4 probes is suggested as the minimum number of probes in a segment that are required to make a call Threshold method and threshold factor This is the value that the algorithm uses to set the threshold value User definable Type the value of the logs ratio into the threshold factor box to set the threshold value that will be used to make a call Typically a logs ratio of 0 5 to 0 85 is used 990055 Version 11 115 Standard deviation the software calculates the standard deviation of the ratio the threshold value will be the threshold factor x standard deviation of the ratios The preferred factor is 3 or 4 Deviation Log Ratio DLR the software calculates the DLR Spread of the ratio the threshold value will be the threshold factor x DLR of the ratios This typically is a factor of 3 or 4 X separation The software uses the value from the X chromosome probes This assumes that a sex mismatch experiment has been run It may omit duplications which usually need a lower threshold value compared to deletions Chromosome average method The method of averaging the segments at the baseline This is used to set the threshold value Median segment uses the Median value of all segments in the chromosome as the baseline and is the preferred option Mean segment uses the Mean value of all segments in t
110. ss frequency in the selected genomic region Mean gain frequency in the selected genomic region 10 Identity of the second selected group 11 P value for aberration frequency in the selected genomic region compared to a second selected group 11 P value for a loss frequency in the selected genomic region compared to a second selected group 12 P value for a gain frequency in the selected genomic region compared to a second selected group OONOOaRWND 990055 Version 11 109 9 Setting up a protocol in Workflow mode CytoSure now has workflow functionality This means that all the various settings for carrying out an analysis are saved in advance as a protocol It is anticipated that a fully trained expert user would set up these settings to form a protocol Once the protocols are created routine users can then follow a predefined workflow using the appropriate settings A description about how to use the software in workflow mode is given in Section 10 All the settings for setting up a protocol are located at Tools Options There are various tabs a description of the options is given here Display lt Options Report Generation Database Files Protocols Normalisation Probe Filtering CBS Annotation Scatter Plot Display Automatic Signal Axis Rescaling Data Point Size Orientation Vertical Signal Axis C Horizontal Signal Axis Karyotype Band Rendering Shading Colour Scheme Col
111. ssfied 25 V9 o 0 Variation 77 FMR2 deleti OPNILW A AL445258 4 6922661 j 986220 pi3 2 0 063 Unclassified 100 L fo Q9 jWaraion 34 45554717 M7735531 q11 21 q11 222 18_ Unclassified 1009 1005 0 f Variation 29 fS rRNA F 196904349 196942211 g29 0 037 Unclassified 100v 1005 Q Q9 Wariation 43 _ MUC20 AC 8229 9051411 _ p22 33 p22 31 9 048 Unclassified 65 46 Oo 9e Variation 77 SHOX Kalm ARSE XG 5 RP11 S85K1 B1874642 31980876 pis i 106 Undassified_ o o Qe fo 7256229 pi17330 231 0 861 Unclassified 100v 1005 0 f Variation 36 DEFE4 DEFBIO4B 21444431 1501132 pi22 0 086 Unclassified 1009 1005 0 Q9 Variation ste JACO096322 69057735 69165872 g132 0 109 Undassfied 1009 1005 0 f WariationA3e UG T2BI7 39356595 99505315 pi1 23 011 20 148 Unclassified 100v 100 0 9e Variation 37 JACIO601133 ogt Once the details are added for all the relevant samples click on the Population Analysis tab The first step is to subdivide the data into autistic samples and n
112. tains box and press Apply Edit Phenotype Details filters Contains 3 Allocate a unique name to this group For this example Autism is appropriate 4 A box appears prompting Would you like to add the opposite group The opposite group will be all those samples whose Phenotype details do not contain the word autism Therefore these could be the normal population Add opposite group o Click Yes to add the opposite group non autism 6 Allocate a unique name to this group Genomic View Table View Aberrations Database Management Population Analysis Chromosome 15 Options NUM 22 NICTENIN q22 L qB g ES o MESS ox HRZ 053 9112 JE 413 19 2 CNN q15 1 1 1 1 L uiu a DEED a Q Gains 15 0 Losses 10 0 Pixels per sample 22 5 0 1 Groups Chromosome Section Combine existing 18 188457Mb 19 491343Mb 20 794230Mb 22 097117Mb autism 20 0 q11 2 x All Aberrations 58 175 ins 24 NOT autism LJ 15 0 Losses 232 All Aberrations 354 12 5 i 122 Venn Diagram autism NOT autism Prader Willi syng Prader Willi Syndrome Type 2 n sT e IBI ena ET B NH P UE Tl 11 RT vd iati ca A 5 r p Pat Patient Figure 93 Plot of aberration frequency 990055 Version 11 104 ogt 8 2 Combining groups Question 2 Combining groups For this question the same example dataset is used as above autism and not autism The software has the abilit
113. tered This is automatically entered automatically Location Reports 96 of the aberration that is annotated as a CNV Does not take into account the DGV classification of whether the CNV is a gain or loss Reports of the aberration that is annotated as a CNV taking into account the DGV classification of whether the CNV is a gain or a loss DB CNV Reports 96 of the aberration that is annotated as a CNV in the internal database Does not take into account the database classification of whether the CNV is a gain or loss DB Gain Loss Reports 96 of the aberration that is annotated as a CNV in the internal CNV database taking into account the database classification of whether the CNV is a gain or loss This reports the CNV regions covered by the aberration It does take into account that the CNV in the DGV database is a gain or a loss If DGV does not report if it the CNV is a gain or a loss then the CNV is reported Reports if the aberration overlaps with the decipher database go to https decipher sanger ac uk for updated Decipher information Highlights human disease genes Note that this list of human disease genes genes is not exhaustive Displays whether the detection was by CBS automatic or manual If for example an aberration was merged Level Automatically generated See section 5 8 for a discussion of the P value User editable See section below Image Location Classification The user can classify the aberration as fo
114. tware LH Genomic View Table View Aberrations Database Management JUs workspace OGT Software db_test ml C Table View Figure 81 The aberrations displayed as an ideogram 990055 Version 11 92 ogt For further detail click on a relevant chromosome and the individual chromosome will be displayed with all the aberrations stored within the database displayed see figure 82 The display can be returned to the whole genome view by clicking on the chromosome CytoSure Analysis Software File View Tools Help D08 1539 A txt ll Invert Raw Normalised Smoothed Display Filtered e S o a Genomic View Table View Aberrations Database Management C Program Files CytoSure Table View Group data by Submission Aberration Whole L Genome Figure 82 Individual chromosome view 7 3 Deleting aberrations in the internal database To delete a relevant aberration highlight the relevant data entry and click on the delete key which will bring up a warning see figure below In order to delete the aberration the commit button at the top right of screen needs to be clicked 990055 Version 11 93 ogt Genomic View Table View Aberrations Database Management C Program Files CytoSure Group data by Submission Aberration Submissions Selected Aberration Aberration Details d p b em mnia ueri eile ia i e s li Delete
115. umes that a sex mismatch experiment has been run It may omit duplications which usually need a lower threshold value compared to deletions Include X chromosome Choosing False will exclude the X chromosome Include Y chromosome Choosing False will exclude the Y chromosome probes This might be used when Female sample and references are used Chromosome average method The method of averaging the segments at the baseline This is used to set the threshold value Median segment uses the Median value of all segments in the chromosome as the baseline and is the preferred option Mean segment uses the Mean value of all segments in the chromosome as the baseline This could be affected by large aberrant regions in the chromosome Zero will set the baseline at zero whatever the data is 990055 Version 11 51 Chromosome Mean log probes Uses the mean value of all the probes as the baseline Chromosome Median log probes Uses the median value of all probes as the baseline Click done The CBS will start running segmenting the data The number of threads can be increased The more threads used the faster the processing however alternative processes on the computer will be slower A quad computer using three threads should take approximately 4 5 minutes to process data from a Syndrome Plus 2x105k array 7 When the CBS is complete horizontal lines will be drawn representing the segments 8 Aberrations called are represented by shaded
116. ustomising the classification terms Customising classification terms allows the user to define their own classification terms Select Tools Options Classification 990055 Version 11 66 Options Normalisation Probe Filtering CBS Annotation Report Generation Database Files Protocols Add new classification Name Colour m Add Classification Remove Selecked Existing classifications Colour de nova parental origin of rearrangement undefined de nova arising on maternal chromosome e novo arising on paternal chromosome Familial inherited From normal mother and Father Apply Changes and Close Figure 57 Adding custom classification The user can type in the name of the classification in the relevant box and then click on the colour box to change the colour On clicking Add Classification a new classification term will appear This new Classification term can then be used in the Aberration table 990055 Version 11 67 Options ES isola enome Normalisation Probe Filtering CBS Annotation Classification Report Generation Database Files Protocols Add new classification Mame DL NV Colour S Existing classifications Colour e nove parental origin of rearrangement undefined e nove arising on maternal chromosome e nova arising on paternal chromosome Apply Changes and Close Figure 58 Adding custom classification The results of a custom class
117. w Table View Aberrations Database Management Population Analysis Chromosome 3 Normalised Smoothed Display Filtered ogt Sample Information S Sample ID eS N E Experiment Sample M vs Reference M N 3 S N m E Sample Details o 2 ur Chromosome Section e 4A F 164 042738Mb 164 068009Mb 164 093281Mb 164 118553Mb JR 29 EET Phenotype Details E a p S 3 Inv 11 212 E i EP 13 3 14 v N s s b N lt gt Uu N 15 a 16 Aberrations D F 1 2 Chr Start Stop Mb s e 17 E 18 3 164067097 164093831 0 02 5 3 hy NAT F L j 19 20 Inv K am N as m N N 10 probes displayed O 111 1 Variation 4360 BE paien OO O O O OO OOOO O Bl edia S RI CNVR1608 1 E oc x m um lt Whole Genome Figure 48 Adding an aberration to the list 5 4 Link to SUSPECTS A link to SUSPECTS has been added in the genomic view SUSPECTS is a site that uses publication information and theoretical calculations such as gene expression data to highlight genes in an aberration that may be disease causing For more details visit the SUSPECTS website In genomic view with an aberration present right click on the aberration and select SUSPECTS 990055 Version 11 57 ogt Genome Genomic View Table View Aberrations Database Management 2 1 1 Y EAA ROE Eaa erum LAT Fp inni EAE
118. window size in the dialog box that appears and click Smooth A larger window size will result in a smoother line through the data The window size number is in base pairs Once the software has calculated the smoothing line click on the Smoothed radio button to display the Smoothed line CytoSure Viewer File View Tools Help Raw Mormalised De Noised Smoothed e LJ o Chromosome 5 x Separation File 1 0 7819035660411758 Genome Chr Start Stop 2 IS l E MIELE T 51 tt I in E AT U L 1 Chromosome Section n 60 613586Mb 67 494056Mb 74 374520Mb 81 254383Mb Hin 1 ran La FIIIT TIE E LH E au kal mh H mmi E EN E ss TOTT N e mg N kal LEE M hJ N 438 probes displayed Figure 28 Smoothed normalised data 990055 Version 11 38 Oxford Gene Technology ogt 4 7 Multiple datasets It is possible to compare two or more datasets on the same plot by importing additional feature extracted files CytoSure Interpret File view Tools Help Eh D Load E Genome Genomic View Table View Aberrations Database Management Population Analysis Chromosome 8 Display Filtered v Normalised Smoothed _ Sample ID l 1 Experiment Sample M vs Reference M dE we uie p PUE Se JA Sample Details Chromosome Section 39 007163Mb C T T oootezzszsz Phenotype Details L K amp 3
119. xis It might be necessary to first turnoff automatic rescaling see section above Genomic View Table View Aberrations Database Management Chromosome 17 Figure 32 Display with smoothing line added h i x x x Chromosome Section Changing ideogram view 13 799906Mb 17 113169Mb T 20 4286432Mb S ule piz p11 2 g11 1 g11 2 To alter the shaded view of the chromosomes to a 2D view click Tools Options Karyotype Band Rendering 43 probes displayed Sl 3 777 NNI GLIM M l METUIT MINIM INEI LT T OHIO N Rl LL WI PP 1 T 111 0 SB TRINH I D LI IL I i Lp DET HT PIT 990055 Version 11 42 Options ass x sto Report Gener ation Database Fies Protocols Display Genome Normalisation Probe Fitering Aberration Detection Annotation Scatter Plot Display 7 Automatic Signal Axis Rescaling Data Point Sce 3 gt Colour 1 I ese Colour 2 B to Colour 3 ee Figure 33 Altering the Karyotype Band Rendering Genomic View Table view Aberrations Database Management C Program Flesh Table View Ideogram View QC Trends Group data by Submission Aberration 1 14 990055 Version 11 Com 43 Figure 34 3D chromosomal ideogram Genomic View Table View Aberrations Database Management C Program Files O Table View Ideogram View O QC Trends Group data by Submiss
120. y to determine the aberration frequency in autistic samples using different populations See illustration below In this example a population identifier Latvia has been added to the sample details Genomic View Table View Aberrations Database Management Population Analysis C Program Files Cy Table View Ideogram View O QC Trends Group data by Submission Aberration Refresh Commit H Backup J petore Export Submissions Scan Date x Aber s Submissi 7 File Location Array Bar C Array D v Sample ID vy Sample vy Referen v Samp 7 Phenoty MAC Ad 7 UserName QC Metrics CBS Calling 12 10 2008 0 4 Wed May 06 1 P test XDBIL WENN eeeeeeeeee D0 1A A0 C8 scan DLRSPREADz CHROMOSOM lis Tue Mar 24 09 P testiXDBIL Syndrome Plu autism Latvian 00 1A A0 C8 scan CHROMOSOM 11 03 2008 1 5 Thu Mar 26 10 S softwarelC Syndrome Plu autism Latvian D0 1A A0 C8 scan CHROMOSOM 12 12 2008 0 112 Fri Dec 12 15 P 0184 cghid Latvian 00 1A A0 C8 scan CHROMOSOM 08 14 2008 1 12 Fri Dec 12 15 PAtestlXDEAL Syndrome Plu Latvian 00 1A A0 C8 scan CHROMOSOM 08 14 2008 1 13 Fri Jan 02 12 IPAtestXDEAL Syndrome Plu Latvian 00 14 40 C8 scan CHROMOSOM 08 14 2008 1 20 Thu Mar 26 11 P AtestYXDBIAL Sy

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