What`s new SEQUENCE Pilot
Contents
1. 15 6 5 Operations cece eese cess ceseeeeeeeseeeseeeseeseeeeeseeeeseegeees 15 re ey statin easiness ied ee dain se eel 16 00 1 Section IER pct sects MM MUR 16 6 6 2 Section Files Info window Files context menu item show lnfo 16 GOS C RR 16 Vallaton MUON TADIC DTI TERNOS 16 CES ee de a RR RR 17 aW Ei iis e RRNNERP 17 17 EXIIT 18 7 Module aia PODER b HERR ERENT DIM EOD 19 SA ee BINE ESO OT PR PEUT mme 19 What s new SEQUENCE Pilot 4 1 1 2 1 All Modules 1 1 All tables Each user can decide which table columns are shown Therefore right click a table header Then select Manage table columns A dialogue opens which contains the following information A list of invisible2. MERIT mm 5 22 Me 2 moder diet ena ean eT ae me MEM M MEM 5 SIS Sie ib eee are TNNT 5 c iR TU TU UU MITTIT 6 SNB ees E IS RR 6 02 FOL Master Me RESTE 6 ra EE E nee EO 6 RU T ERE AE EE 7 SCA NEC Ec MENTRE PTS 7 DEEE 5 7 0223 lab Chromosome 5 T ET 7 6 2 3 New tab add ERPCOGDITIBITUI IL 8 6 3 Operation ROI Groups master TG i 9 SR ME NIS SEIT 9 fom 9 BAL TAE aus eee ers 10 Tap rUe MOO eor 13 eei TT ENEE 13 DATA Tab BAN SAN 55 14 TERS ISD a E EEEE 14 6 4 1 6 Tab
3. 5 Sorting Distinct other cov Distinct Homop cov gt thresholds thresholds Distinct mutations Other homopolymer mutations 6 4 1 2 Tab Hide Mutation Here settings for special mutation that should not be called can be set If one of these items is checked corresponding mutations are not listed in the Variation Mutation table e without WebRef e Without MutDB entry e single direction reads mutations that are present due to the activated settings Single double direction analysis marked pink in the Variation Mutation table are not shown e CNV mutations from CNV analysis e Silent mutations e intronic mutations e frameshift mutations e splicing mutations 6 4 1 3 Tab Quality Score Here settings to exclude bases with bad quality from analysis can be set e Quality score threshold What s new SEQUENCE Pilot 4 1 1 13 o case there is a value entered e g 15 only bases with a quality score above this value are called Only called positions are counted to the coverage o The quality score filter can be switched off by selecting Quality score threshold off o n the operation Sequence base positions that were not called due to bad quality are shown as bases but are greyed out In the tool tip of the forward reverse and combined sequence there is a new entry Ns Ignored showing how many bases at a position were ignored due to bad quality In the coverage graph the coverage of bases that were n
4. Optionally enter the number of the column containing the antisense primer It is also possible to enter a default cutting length The number of bases entered here are then removed from each amplicon What s new SEQUENCE Pilot 4 1 1 8 Press OK after the fields are adapted Moreover the creation of ROIs out of manifest files has been improved Primer pairs are loaded and considered in the field Amplicons PCR Primers 6 3 Operation ROI Groups master file The new buttons Export Import Save as and Import Primer are available Export lmport Groups be exported and imported using the buttons Export and Import respectively For the ROIs included the group all information given in the operation RO master file Ignored Parts Pseudogenes Amplicons and Primers and CNV analysis modes are also exported and imported Moreover you are asked if you want to export an existing profile defined in the operation Run as well Therefore you can select a profile to export with the ROI Group Save as Saves copy of the selected ROI Group You can enter a new ROI Group name and lot number Import Primer For a selected ROI Group file containing a list of primer pairs can be added The primers are joined to the corresponding ROI automatically In the operation RO master file they are shown in the section Amplicons PCR Primers During import the window Import Primer CSV Txt Fi
5. 3 Choose this setting in case barcodes have to be present at both ends of the reads o ignore paired end info checked paired end information is not used e Mutation table Warning Percentage of bases of a read that must be not aligned to the reference before the warning W appears in the Variation Mutation table o gap SNP to SNP The maximum number of bases between two base changes to consider those two base changes as an InDel o Indel gap SNP to InDel The maximum number of bases between base change and an InDel to consider them as an InDel 6 5 Operations Joining Worklist Poollist In the search field Gene all genes with defined ROls in the operation RO master file are available In older versions only genes that were defined in the 1is ini file were displayed If a gene is selected in the search field Gene only orders including this gene are shown in the Lower table When orders are opened the sequences of the selected gene is shown automatically When What s new SEQUENCE Pilot 4 1 1 15 you jump to previous or next order using the buttons Previous Next respectively the gene is selected as well 6 6 Operation Sequence 6 6 1 Section Order Settings MIDs are shown behind the Barcode in parenthesis This also works for old orders analysed with version 4 0 1 or previous 6 6 2 Section Files Info window Files context menu item show gt nfo MIDs are shown behind the Barcode Barcode
6. The new tab is available which shows information about the ROI This tab was named Amplicon in older versions On tab the average coverage fwd rev is listed for the complete ROI Moreover the number of assigned reads trimmed reads and aligned reads is listed in the corresponding columns The tab Amplicon now corresponds to the defined amplicons in case an ROI was sequenced using several amplicons There are only entries present for ROIs with defined amplicons Here the average coverage fwd rev for each amplicon is shown Moreover the number of aligned reads is listed in the corresponding column 6 6 6 Show There is the new jumper ow quality available to jump to positions with low quality Those positions are present in case the setting Low Quality score coverage warning operation Run is exceeded In this case there is also the warning low quality in the column Coverage of the RO s Locations table 6 6 7 Electropherogram The user can decide to show either one electropherogram for the combined sequence or two electropherograms for forward and reverse sequence separated Therefore the new combo box combined fwd rev is available below the count mode combo box Min coverage line For orders analysed with software version 3 5 2 and older the value for the Min coverage line is O therefore the default settings of the 1is ini file is used as Min coverage line The context menu item show fragments view available in combined forwa
7. in which column of the file the necessary information locates Therefore also self made csv files containing ROI information can be loaded now Press the button Columns in the section Settings The new window mport Bed Text Files opens The settings entered here already are the default settings for usual bed files They only to be changed according to the layout of the bed file you are using The following fields can be adapted Separator Here the column separator TAB or be selected Ignore Header If this is active the first line is regarded as header and therefore ignored Column positions o Chr Enter the number of the column containing the chromosome number o Chr Start Enter the number of the column containing the chromosome start o Chr End Enter the number of the column containing the chromosome end o Gene Optionally enter the number of the column containing the gene name o Transcript ID Optionally enter the number of the column containing the transcript ID Comment Optionally enter the number of the column containing a comment Chromosome positions include primers Check this option in case the chromosome positions include primers Amplicon PCR Primer o Sense Primer Optionally enter the number of the column containing the sense primer It is also possible to enter a default cutting length The number of bases entered here are then removed from each amplicon o Antisense Primer
8. new the context menu item filter This serves to get an impression on how the Variation Mutation table changes when other settings are used This function helps to optimize you settings used for the run In the Filter window the Settings used in the operation Run on tab Settings and tab Hide Mutations can be changed Not all settings can be changed it is only possible to change settings that reduce the number of mutations listed in the Variation Mutation table The section Variations shows the number of variations mutations listed on the five tabs of the Variation Mutation table when the settings shown above are applied When OK is pressed the new settings are applied All variations mutations listed in the Variation Mutation table are then sorted to the tabs regarding the new settings Tab Filter now lists all variation mutations that do not pass the filter settings and would not be detected in case the run was recalculated using the filter settings When the operation Sequence is left the filter is reset and all variations mutations are re sorted to the tabs they have been listed on before In the Filter window a setting can be saved as a profile by pressing the button Save Profile This profile can then be used to recalculate the file to apply the new settings 6 6 5 Summary The seach function is not available any more On all tabs the new column ndex is available In this columns the entries are numbered starting from 1
9. pressing Import the SNPs are imported into SEQUENCE Pilot This may take up to four hours A status bar is shown during this time 2 2 Gene Admin The following changes are present In the isoform table there is the new column 7 available to index each isoform For SeqPatient only The number listed here is the same number that is shown in the operation Sequence written in parenthesis in PCR Product Location table in case several active isoforms are used to analyse a file For the first active isoform it is checked if there are several exons with the same name present If this is the case the user will get a warning There is the new button renumber exon names available in the dialogue Change Gene Using this exons are renumbered starting from E1 for the first exon to EX It is possible to map genes downloaded with Gene Admin to the genome hg 19 Advantages are that the genome position is then shown in the Variation Mutation table column Pos Moreover imported SNP databases see previous chapter are then applied Therefore the new context menu items Extras gt gt map to genome hg19 gt be used o Selected gene the gene selected in the Genes table is mapped to the genome hg19 all current genes all currently used genes genefile selected in the Versions table mapped to the genome hg19 Please note that this can take several hours depending on how many genes are present in Gene Admin You will therefor
10. result is visualized in a window next to the table it is reported if all ambiguities can be resolved or not 3S Primers and or additional exon primers are listed resolved ambiguities are listed beneath the primers unresolved ambiguities are listed for result calculation 6 digit resolution is used What s new SEQUENCE Pilot 4 1 1 5 6 Module SeqNext 6 1 Multiplicom MASTR assays Multiplicom MASTR assays for copy number variation analysis Definition files for easy set up of ROIs for Multiplicom MASTR assays including copy number variation CNV analysis are now also available To obtain these please contact Multiplicom at customerservice multiplicom com specifying the MASTR assay and SeqNext as target software After importing the ROIs the following has to be set up for the CNV analysis For more details see User Manual SeqNext e all ROIs belonging to the MASTR assays have to be grouped in the operation master file a CNV analysis mode has to be defined for the ROI Group in the operation Analysis Mode CNV master file Here all control ROIs have to be defined as control C and all other ROIs as targets T 6 2 Operation ROI master file 6 2 1 All Tabs Note For old ROIs including PCR Primers the PCR Primers have to be entered again The term amplicon part was replaced by amplicon Amplicon now refers to the reads blocks present in case a large ROI was amplified using several primer pairs The field
11. 3 New tab add Enrichment Kit The tabs add Enrichment and add Kit were combined into the tab add Enrichment kit Using the radio button Enrichment or Kit you can select if you want to set up ROIs for an enrichment or a Multiplicom MASTR assay Select transcripts to build ROIs For enrichments as well as for Multiplicom MASTR assays it is now possible to select transcripts for each gene After build is pressed the new window Change Gene Transcript opens automatically In this window all genes defined in the enrichment Multiplicom file are sorted by Chromosomes and Chromosome Range For the selected chromosome all automatically build ROls and used transcipts are listed in the RO s table Above the tables the chromosome range of the selected gene and all available transcripts are shown graphically The ROI positions defined in the file are indicated as green bars The transcript automatically used to build the ROIs is marked red For selection of this transcript SeqNext chooses the one where most of the ROIs defined in the files cover exonic regions In case there are several transcripts present that are equal in this matter one of them is choosen randomly Another transcript can be selected by right clicking on it and selecting set to transcript The ROIs are changed automatically using the new transcript When all transcripts are the desired ones you can press OK to close the window Add Enrichments For csv or bed files it can be defined
12. 9 should be used for mapping this has to be set in the lis ini file located in the bin directory of your installation Enter the directory name of the genome behind the entry DefaultGenomeDir in the section SeqPilot e g DefaultGenomeDir HomoSapiens This genome directory has to be present in the folder Genefiles Genome of your installation Note JSI offers an exe file to import the SNP databases snp135 and snp137 from NCBI into SEQUENCE Pilot To receive this please contact our support team Alternatively you can import any other SNP database doing the following SNP database files be downloaded for example using the following links o Ensembl in GVF format http www ensembl org info data ftp index html o NCBI in txt format htto hgdownload cse ucsc edu goldenPath hg19 database e g Smnpl27 bxb oz The downloaded file has to be extracted in the new directory GeneFiles GenomeVar directory of yur SEQUENCE Pilot installation What s new SEQUENCE Pilot 4 1 1 3 import the SNP database do the following after selecting the menu item mport SNP DB file o Select the genome the SNP database refers to in the field Organism e g hg19 o Then press and select the downloaded SNP database file it locates in the GeneFiles GenomeVar directory o n the field Version information for identification of the SNP database file such as genome date and name of the SNP database file can be entered
13. Amplification was renamed into Category The function stays the same The column Extended was renamed into Reference The new column ndex is available to index the table entries In case the location of an ROI is changed column Location of the ROI List defined amplicons for the ROI are adapted automatically ROIs can be exported or imported using the buttons Export and Import respectively Using the export function all ROIs listed in the RO List are exported Information such as amplicons primers ignored parts and pseudogenes are also exported imported The fields Amplicons and PCR Primers were merged into the new field Amplicons PCR Primers Amplicon definition By selecting an ROI in the list and pressing Add amplicons can be entered as done before Primer definition Primer pairs can be added for a selected ROI by pressing Add These primer pairs then define the borders of the amplicons primers are not included Amplicons are therefore created automatically when a primer pair is entered For primers an 5 and 3 error rate can be entered Amplicons PCR Primers can be inactivated by removing the mark in the column Active Inactive amplicons are not used Moreover amplicons can be defined for both forward and reverse reads or for forward and reverse reads separated by selecting the corresponding entry in the column Direction Moreover a csv txt file containing a list of primer pairs be imported by pressing Imp
14. Ext in parenthesis Old orders analysed with version 4 0 1 or previous have to be recalculated to show MIDs All settings used for the Run are listed The section RO showing the number of aligned reads is not available any more in the nfo window These informations are now shown using the context menu item show gt info the ROl Location table or using the Summary button 6 6 3 Section ROI Location table The new context menu item show gt info is available now the number of assigned and aligned reads for the selected ROI are shown Moreover the number of trimmed reads is displayed The ROI Info window context menu item ROI info has two tabs now Future analysis and Current analysis On tab Current analysis information about the ROI used for the current run are shown ROIs be changed on tab Future analysis only In case the ROIs are changed the ROls that will be used for future runs are shown on tab Future analysis Changes such as new Amplicons Ignored Parts or Ignored Sequences are highlighted After doing a recalculate the highlighting disappears and the new ROI information are also shown on tab Current analysis In the column Coverage there is the new warning ow quality available This is shown in case the setting Low Quality score coverage warning operation Run is exceeded You can jump to positions with low quality using the jumper ow quality in the Show window 6 6 4 Variation Mutation table For
15. Ols completely covered by both sequencing directions If this is the case switch the two Both direction coverage settings to off the ROIs partly covered by one sequencing direction only single and partly covered by both sequencing directions double ROI forward reverse single double single In case you want to call mutations in the single regions fill out at least on of the Both direction min 96 coverage settings The settings Both directions min abs coverage and Both directions min coverage define if the position is covered by a single direction or by double both directions For double direction regions all other settings entered on tab Settings are used as they are entered For single directions some of the other settings are changed to the combined mode o Ratio read directions for mutations and reads are switched off o Min abs coverage for reads and mutations Min coverage Distinct Other coverage Distinct Homopolymer coverage are set to combined This way mutations detected in single direction regions can be called They are marked pink in the Variation Mutation table whereas mutations detected in double direction regions are not marked o Both directions min abs coverage If the coverage is above the here entered value in both sequencing directions we have double directions If the coverage one sequencing direction is below or equal the here entered value we have a
16. Whats new SEQUENCE Pilot Version 4 1 1 03 19 2013 SEQUENCE 19 h Pilot medical systems EB HE EH HEN GmbH aH www jsi medisys com Am i PHAN ye 7 ie pr ort Laer pp OF T CARRA ANDET A DU ao a a 1 E 1 1 ee 1 1 Doi developed JSI medical systems GmbH JSI medical systems Corp Friedhofstr 5 1901 Newport Blvd Suite 350 77971 Kippenheim Costa Mesa CA 92627 GERMANY USA phone 49 7825 863620 0 phone 1 949 999 2092 fax 49 7825 863620 20 fax 1 949 999 2093 email mail gjsi medisys com email mail us jsi medisys com web www jsi medisys com for research use only Table of Contents UE ROREM POR 3 UEM mme 3 2 Modules SeqPatient SeqC 3 2 1 Menu SeqNext SeqPatient and 5 3 GB C ANN NUT NETTE 4 DEL knHYEHPRORRERFANSEEPA HIERO 4 Aal Isid cU CBS CRIT m 4 Es ag carcasses ene UEM MM vee tne eee 5 2 Modules SeqPatient and 5 Jomning 9 4 Module lari BNET TT TT NEN 5 EE ceo rMRRRRRURDRS H 9
17. ation table The settings on this tab are important to adapt for your analysis The first setting 1 Single double direction analysis is explained in the end of this chapter because if not switched to off it can change the other settings Reads The settings entered here are applied to all reads 2 Analyse ignore region A region is not analysed no variant calling in case these settings are not fullfilled Please note that the settings may be changed automatically in case the single double direction analysis is activated o Min abs coverage Positions with an absolute coverage below this value are written in grey mutations are not called In the combo box behind you can select if the Min abs coverage has to be reached in both sequencing directions together select combined or in each direction separated select per dir Example Min abs coverage is 20 combined If there is a heterozygous position with an absolute coverage of 19 the mutation is not called o Ratio read directions This value is the ratio between the two read directions forward and reverse Positions where the ratio is not fulfilled are written in grey mutations are not called Examples read directions is 5 05 If there is a position with 100 reads 4 reverse and 96 forward reads the ratio is 4 96 The position is greyed out a mutation at this position would not be called If switched to off mutation calling in regions covered by o
18. ched for also in reverse complement in all reads Enter an adapter sequence in 5 gt 3 direction The following fields can be edited for each entered adapter o Position auto It is decided automatically if the adaptor locates at the 5 or 3 end 5 Adapter be found at the 5 end only 3 Adapter be found at the 3 end only o Error rate here a percentage value can be entered as error rate wrong bases that the adapter can contain o Overlap Here the minimum number of adapter bases that must overlap with the read can be entered Example overlap is 3 There must be at least 3 adapter bases found in the read Remove ends This function allows to automatically remove sequences at the ends of the reads e g adapters that do not match to the reference The user can decide to remove bases at the 5 and or 3 end by activating the box For removing ends a number of bases beginning from the end of the read to the number given in the field Distance is checked o Incase the are no mismatches nothing is removed o Incase there are mismatches it is checked if sequences start to match within the Distance What s new SEQUENCE Pilot 4 1 1 14 n case match is found the matching sequence must have at least the length entered in the field Blocklength It can include as many mismatches as entered in the box Max mismatches The bases before for 5 or behind for 3 the first matching base are then remo
19. columns on the left side A list of visible columns and their width on the right side A header preview in the lower section You can remove or add columns by moving them into the corresponding list Therefore press Add or Remove or double click an entry Moreover the column width can be changed in the list of visible columns by entering a number or by changing the preview below In both cases the preview is adapted By pressing Default the default settings including all columns are applied By pressing OK the new settings are applied The settings are saved for each user Note In case the table is exported only visible columns are exported 2 Modules SegqPatient SeqC and SeqNext 2 1 Menu SeqNext SeqPatient and SeqC The new menu item mport SNP DB file is available Using this SNP databases can be imported into SEQUENCE Pilot For the import of SNP database files it is required that the genome the SNP database refers to e g hg19 is present in the SEQUENCE Pilot installation directory Genef iles Genome hg19 For instructions how to install hg19 please check the User Manual SeqNext or contact our support team After import For all ROIs created using the corresponding genome e g hg19 the SNPs are shown automatically Genes downloaded using Gene Admin have to be mapped to 979 first before SNPs are shown Gene Admin Extras gt map to genome hg19 see next chapter Note In case another genome not hg1
20. e following colors are used by default e quality score 1 10 Dark Red quality score 11 20 Red quality score 21 30 Yellow quality score 31 40 Light Green quality score 41 99 Dark Green Original reads The original length of the reads is shown when the field org reads is checked The minimum and maximum number of bases that were removed left and right is indicated in color To see the sequence of the removed bases the new context menu item show original reads is available When this item is selected a new reads view opens showing original reads Bases that were removed at the left and the right a highlighted The read identifier is shown in the tool tip of the read when the box org reads is checked Here the file and the line which the read can be found is shown The read identifier is also shown as a tooltip when the new context menu show original reads is used For paired end sequencing data the paired end key is shown in this tooltip as well New button Search is available Using this you can either search for a sequence in the reads or for read by giving the line number of the Next Generation Sequencing file The Search window opens when Search is pressed To search for a sequence o Enter a sequence to be searched for in the field Sequence o After pressing Search all reads containing this sequence are listed a new window sequence that was searched for is highlighted To search for a read listed
21. e get a warning o all current history genes all downloaded gene versions including all genefiles shown in the Version table are mapped to the genome Ag19 Please note that this can take several hours depending on how many genes are present in Gene Admin You will therefore get a warning For using this item it is required that the genome hg79 is present the SEQUENCE Pilot installation directory Genefiles Genome hg19 For instructions how to install 979 please check the User Manual SeqNext or contact our support team 2 3 Operation Sequence Chip 2 3 1 Variation Mutation table The following table columns changed New column ndex Here the table entries are indexed New column HGVS nomenclature Here the HGVS nomenclature for each table entry is listed The column mut Ref does not exist any more In case the mutation is present in the internal mutation database the column mut Entry shows the frequency homo hetero of the mutation In case the Nuc Name was changed in the mutation database and differs from the entry in the column HGVS nomenclature it is listed in brackets What s new SEQUENCE Pilot 4 1 1 4 2 3 2 entry of the column mut Entry frequency of a mutation can be listed the report 3 Modules SeqPatient and SeqHLA 3 1 Operations Joining and Worklist In the field Validation the new search item required is available to search for orders with open requirements 4 Module SeqPat
22. f the ratio is not fulfilled mutations variations are not called Examples Ratio read directions is 5 95 If there is a mutation detected in 100 reads and there are 4 reverse and 96 forward reads showing the mutation the ratio is not fulfilled The mutation is not called If switched to off mutation calling in regions covered by one sequencing direction only is possible o Min coverage If the 9o coverage of a mutation is below this value the mutation will not be called You can choose if the Min 96 coverage should be calculated per direction or combined combined The coverages in forward and reverse sense are calculated combined coverage total coverage of the mutation total coverage at the position dir The coverages in forward and reverse sense are calculated separated Both values have to reach the Min coverage forward coverage forward coverage of the mutation total forward coverage reverse coverage reverse coverage of the mutation total reverse coverage 5 Mutations Sorting The values entered here decide if a mutation is listed on tab distinct other or homopolymer of the Variation Mutation table Please note that the settings may be changed automatically in case the single double direction analysis is activated o Distinct Other coverage f the coverage of a mutation reaches this value the mutation is listed on tab distinct of the Variation Mutation table Otherwise it is
23. has the following formate e g 100 200 shows that only the bases 100 to 200 of the asta sequence are used as ROI The number entered here is listed in the field Location of the ROI list 6 2 2 3 Tab Chromosome Range This tab has been worked over chromosomal positions can now be entered directly into the field Position In case a position is entered here the fields Chromosome and Range are filled out automatically Optionally the position can be entered into the fields Chromosome and Range directly New button go Press this button to start the calculation of the chromosome range The button Add gt in the upper part of the screen adds the complete chromosome range as one ROI to the ROI List Moreover single genes or exons that locate in the chromosome range can be added to the ROI List Therefore you can select a gene a transcript and exons in the corresponding tables The tables are the same as for tab Gene ROIs can be added to the List by selecting one or several exons and pressing Add 1 2 in the lower part of the screen Alternatively Add gt can be used to add several selected exons as single ROIs Optionally a suffix to be added to the RO Name can be entered in the field Suffix What s new SEQUENCE Pilot 4 1 1 7 In the exon table all exons that are completely inside the chromosome range are marked green Exons that do not lie within the chromosomal range are not marked They can be added as well 6 2
24. ient 4 1 Operation Joining In case the lis ini entry forced join is set JoinRFForced yes section SeqPilot the forced join to an order is done in the following cases Result files with defined DNA number and SeqPrimers sample naming that can not be aligned to a gene are forced to join to an order was already available in version 4 0 1 For result files with defined DNA number and SeqPrimers containing less than 50 sequenced bases or entry behind MinRFBases in the lis ini file section SeqPilot no alignment to a gene is done Those files are also forced to join to an order as well 4 2 Operation Sequence In case there is a comment present for a position resultfile there will be the entry in the column Condition of the Position Resultfile table 5 Module SeqHLA 5 1 Operation Sequence In the Matching table tab Total Result the context menu item show sequence specific primers has been completely worked over This should simplify the selection of HLA SSP Sequence Specific Primer Typing Kits from R O S E Europe GmbH to resolve ambiguities In case there are ambiguities present the 3S Primers that are able to resolve the ambiguities are marked automatically If more than one 3S primer is able to resolve an ambiguity all of them are provided to let the user decide which one to use A suggested primer is marked green and required automatically If 3S Primers are not sufficient additionally exon primers are required The
25. in the Next Generation Sequencing File o Select the file in the first field of the section Line o Enter the line number in the second field of the section Line When Show read group is active not only the read present in the entered line but all identical reads are listed o After pressing Search the read s is are listecd in a new window The line information is available in the tooltip of the read when org read is active What s new SEQUENCE Pilot 4 1 1 18 7 Module SeqNext HLA The module SeqHLA 454 was renamed into SeqNext HLA 8 Module Talkmaster Orders with open requirements can be exported in the Joining and Worklist To search for orders with open requirements the new search item required is available in the field Validation of the Joining and Worklist A new tag Confirmation was added to the TalkMaster export Using this confirmed or rejected can be exported What s new SEQUENCE Pilot 4 1 1 19
26. les opens Here you can define the file layout o Separator the column separator TAB can be selected o Ignore Header If this is active the first line is ignored o Column positions Sense Primer enter the number of the column containing the sense primer Antisense Primer enter the number of the column containing the antisense primer 6 4 Operation Run sam and bam files can be analysed now In case you want to load sam or bam files select the reference genome e g hg19 in the field Organism Moreover in the settings chosen for the run there is then new tab bam sam available to select settings for analysis of bam or sam files 6 4 1 Settings The settings in the operation Run changed bam and sam files can be analysed now In case you want to load sam or bam files select the reference genome e g hg19 in the field BAM SAM Organism The settings are sorted on the tabs Settings Hide Mutations Quality Score Trimming BAM SAM and Expert Settings Note The settings on tab Settings are very important to adapt for your analysis A default setting can be chosen that is selected automatically when the operation Run is opened Therefore check the box as default before saving the profile by pressing Save Profile What s new SEQUENCE Pilot 4 1 1 9 6 4 1 1 Tab Settings Here settings can be adapted which influence mutation calling and the sorting of mutations to the different tabs of the Variation Mut
27. listed on tab other o Distinct Homopolymer coverage f the coverage of an insertion deletion in a homopolymer region reaches at least this value the mutation is listed on tab distinct of the Variation Mutation table Otherwise it is listed on tab homopolymer You can choose if the Mutations Sorting coverages should be calculated per direction or combined combined The coverages in forward and reverse sense are calculated combined coverage total coverage of the mutation total coverage at the position dir The coverages in forward and reverse sense are calculated separated Both values have to reach the Mutation sorting coverages forward coverage forward coverage of the mutation total forward coverage reverse coverage reverse coverage of the mutation total reverse coverage 6 Homopolymer o Homopolymer region size The minimum number of repeats of a base to be considered as a homopolymer region Reads include PCR Primer Please fill out the field choose yes or no in case you have amplicons defined since it improves your amplicon assignment o auto please change this setting in case you have amplicons defined o yes select yes in case you reads include PCR primers select no in case your reads do not include PCR primers What s new SEQUENCE Pilot 4 1 1 11 1 Single double direction analysis These settings have to be set according to the data you are analysing Are all R
28. ne sequencing direction only is possible 3 Coverage warning Low abs coverage warning The value entered here is shown as a red dotted line in the electropherogram In case there are positions with a coverage below this value you get a warning for the ROI o There is the hint ow in the column Coverage of the RO Location table o The graph color of the coverage graph below the location overview changes from grey to pink The Low abs coverage warning can be switched off by selecting the entry off If O is selected the default value defined the 1is ini file will be used 4 Mutations The settings entered here are applied only for reads showing a mutation If the settings are not fullfilled the mutation is not called Please note that the settings may be changed automatically in case the single double direction analysis is activated o Min abs coverage If the absolute coverage of the mutation is below this value it is not called In the combo box behind you can select if the Min abs coverage has to be reached in both sequencing directions together select combined or in each direction separated select per dir Example Min abs coverage is 5 combined If there are 4 reads present showing the mutation the mutation is not called What s new SEQUENCE Pilot 4 1 1 10 Ratio read directions This value is the ratio between the two read directions forward reverse of all reads containing the mutation I
29. ort Primer During Import the window mport Primer CSV Txt Files opens Here you can define the file layout What s new SEQUENCE Pilot 4 1 1 6 o Separator Here the column separator TAB can be selected o Ignore Header if this is active the first line is ignored o Column positions Sense Primer enter the number of the column containing the sense primer Anti sense Primer enter the number of the column containing the anti sense primer 6 2 2 Tab add PCR 6 2 2 1 Tab Gene The direction of the gene is shown in the new column Strand in the Genes table In the isoform table the new columns AA length Start codon and Stop codon are available For each transcript the following information is listed AA length Number of amino acids In case the transcript is not translated there is the entry O in the column AA length Start codon Exon number and position of the start codon Stop codon Exon number and the position of the stop codon In the location table the chromosomal location of each exon is shown in the column Position There is the new field Suffix Optionally a suffix can be entered to be added behind the ROI name listed in the field Name The button Add gt was renamed into Add 1 gt 6 2 2 2 Tab Fasta The button Add X 2 is not available any longer The new field Range is available A sequence range to be used as ROI can be entered in case not the complete fasta file should be used The entry
30. ot called due to bad quality is shown in a lighter grey Ignore reads threshold Here a percentage value can be entered If the bases with bad quality in the complete read including primers adapters exceed this value the read is ignored Bad qualtiy means that the quality is below or equal the Quality score threshold setting or that Ns were called by the sequencer Low Quality score coverage warning Here a threshold to get a warning for ROIs with a bad quality score can be set In case bases with low quality exceed the here entered percentage value at a position in the ROI the warning ow quality is shown in the column Coverage of the table You can jump to positions with low quality using the jumper low quality in the section Show 6 4 1 4 Tab BAM SAM sam and bam files can be analysed now Here you can choose the settings used use BAM SAM mapping If this setting is active the BAM SAM mapping is used SEQUENCE Pilot does not do an own mapping use BAM SAM alignment f this setting is active the BAM SAM alignment is used SEQUENCE Pilot does not do an own alignment 6 4 1 5 Tab Trimming Here settings to trim adapters or to automatically remove sequences at the ends of the reads can be entered The settings adaptable here were available in the previous version already Adapter Here adapter sequences can be entered to trim or discard reads e g for Haloplex or Fluidigm The sequence entered here is sear
31. rd and reverse sequence was renamed into show reads In the in the main view as well as the reads view the count mode amplicon was renamed into ROI abs Ignored bases Bases in forward reverse and combined sequence that are excluded from analysis are written in grey Bases are excluded from analysis in case the settings Ratio reads directions What s new SEQUENCE Pilot 4 1 1 17 and Min abs coverage for the Reads entered in operation Run not fulfilled Bases in the reads sequences that are excluded from analysis are greyed out Bases can be excluded from analysis due to bad quality scores depending on your settings in the operation Runltab Quality Score In the tool tip of the forward reverse and combined sequence there is a new entry Ns Ignored showing how many bases at a position were ignored due to bad quality In the coverage graph the coverage of bases that were not called due to bad quality is shown in a lighter grey There is several new information available in the tool tip of forward reverse and combined sequence Coverage This is the number of called bases at a position Reads This is the number of reads covering a position Ns Ignored This is the number of Ns and ignored bases due to bad quality at a position 6 6 8 Reads view New field quality if this field is activated quality scores are shown as a color code below the bases The item replaces the old item bases quality in the combo box Th
32. single direction Example 5 is entered There are 5 forward and 100 reverse reads The region is regarded as a single direction region the settings are changed to the combined mode There 6 forward and 100 reverse reads The region is regarded as a double direction region the settings are not changed Both directions min coverage If the percentage coverage is above the here entered value in both sequencing directions we have double directions If the pecentage coverage of one direction is below or equal this value we have a single direction Example 5 is entered There are 5 forward and 95 reverse reads 5 9o The region is regarded as a single direction region the settings are changed to the combined mode There are 6 forward and 95 reverse reads 5 1 The region is regarded as double direction region the settings are not changed The flow diagram summarizes influence of the settings on the mutation calling What s new SEQUENCE Pilot 4 1 1 12 Single direction Combined mode ratio read Single double dir Both direction coverages directions off yes TRY Double direction per dirs combined 2 Analyse lgnore region Greyed out abs coverage and hase positions Ratio read directions No mutation yes 4 Mutations abs coverage and we Ratio read directions and gt mutation Min coverage Homopolymer region Szen yes
33. the module SeqNext it is possible to set SegPatient requirements for entries of the Variation Mutation table Therefore the new context menu item require SeqPatient primer is available This item opens a new window where SegPrimers to be required are proposed automatically In case a requirement is set there is the entry Q in the column Condition of the Variation Mutation table Moreover a SeqPatient order is created automatically including the required genes positions The name of the SeqPatient order corresponds to the name of the SeqNext order You can jump between SeqNext and SeqPatient by using Extras gt gt Jump to Sequence Moreover it can be entered if a mutation was confirmed with SeqPatient Therefore the new context menu item confirmation is available to show if a mutation was confirmed or rejected with Sanger sequencing case a mutation is confirmed there is in the column Condition in case it is rejected there is a The confirmation can be reset by selecting confirmation gt reset There is the new column ndex to number mutations starting with 1 There is the new tab filter in the Variation Mutation table This tab has only entries in case the filter function of the Variation Mutation table is used It then lists all mutations that do not pass the filter settings and are therefore filtered from the other tabs of the Variation Mutation table What s new SEQUENCE Pilot 4 1 1 16 Mutations be filtered using the
34. ved Incase there is no matching block found nothing is removed Remove bases Removes bases at the beginning and or end of each read Adaptors are removed prior to that 6 4 1 6 Tab Expert Settings Further settings concerning read processing and mutation detection can be adapted on tab Expert Settings On this tab tool tips are available explaining the settings e Base calling o Genome set If set to haploid the basecalling is expected to be homozygous o Unique reads only checked the coverage of identical reads is set to 1 e Read processing Alignment evaluation Skip evaluation If checked no alignment evaluation is done The filters below not used mismatches Filter for mismatches a read can contain compared to the reference The higher the number entered here is the more mismatches are accepted In case there are too many mismatches the read is discarded Min matching bases Percentage of read bases that have to match to the reference In case less bases match the read is discarded Keep strong consensus The percentage of consecutive bases that have to match to the reference without a mismatch between them If this value is reached the settings Max mismatches and Min Matching bases are overruled and the read is aligned This filter is only applied if the read length is above 100 o Compl reads only checked reads that do not cover the complete amplicon are discarded o Barcode 5
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