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What`s new SEQUENCE Pilot

1. What s new SEQUENCE Pilot 4 2 0 35 Please have a look at our User Manual SeqNext for detailed instructions 6 12 7 4 Context menu The context menu item filter was renamed into filter gt settings New context menu item settings gt zygosity gt set to homozygous heterozygous Using this a mutation can be set homozygous heterozygous VCF export context menu export gt tab to VCF additional information is exported as comment o file date date of the export o Source SeqPilot version o mputFileList The path and name of the imported raw data file s o INFO lt ID profile name profile name in case the settings were modified several entries can be present Note The vcr export also works in operation Joining context menu in the Lower Table for one or several selected runs Tab Warning the context menu entry setting gt add tab filter to tab all is available By default the entries listed on tab Warning are not listed on tab all You can change this using this item To remove warnings from tab all use setting gt remove warnings from tab all 6 12 7 5 Table headers The following new table headers are available Weighting Tab the mutation is sorted to distinct other homopolymer warning Moreover there is the entry forced in case the forced combined mode was used The forced combined mode can be used automatically in case the settings Single double direction analysis or forced combined coverage are set
2. quality This is shown in case the setting Low Quality score coverage warning operation Run is exceeded expected The coverage of one or several positions is below the Expected coverage warning Min abs coverage default 100 The Min abs coverage is represented as a red dotted line in the electropherogram Moreover the Min abs coveage is shown in the graphical overview below the location overview The Expected Coverage warning Min abs coverage can be set in your Settings when starting the Run You can jump to the corresponding positions with coverage warnings using the jumper check required expected no call and quality in the section Show Several new context menu entries are available in the RO s section editing gt o reanalyse reanalyses the ROI result is reloaded e recalculate recalculates the ROI using new settings or changed ROI What s new SEQUENCE Pilot 4 2 0 50 o Settings opens the dialogue Settings to change the settings for the selected ROI only After changing the settings the ROI is recalculated automatically Adaptors can not be changed for a single ROI only for the Run Therefore these fields are greyed out e show o RO Info Information about the ROI such as amplicons primers skipped sequences and parts are shown The same fields can be edited in the operation ROI master file The ROI Info window has two tabs Future analysis and Current analysis On tab Current analysis information about the ROI
3. In the section Overall all errors that occurred are explained e g Error 51 reads have a quality score not satisfying the user specified threshold The number of reads filtered for each error is listed You can find a list of all possible errors by pressig the button In the middle a search for unused reads of certain Genes ROIs Error codes or Sequences can be done If the button Search is pressed o the section Searched lists the information about unused reads depending on the search criteria o the table below displays all reads that fullfill the search criteria The following information is given ROI name of the ROI or Homology region the read could be mapped to If the read could not be mapped to an ROI this field stays empty Error Error code number Base seq Base sequence of the read One or several selected base sequences can be copied Therefore do a right click on the sequence and select copy selected base sequence s from the context menu What s new SEQUENCE Pilot 4 2 0 32 6 12 6 Summary All tabs Column Bases is not available anymore new column Cov Info Here coverage warnings not analysed dropout nocall required quality expected for the Gene are listed Note Only one coverage warning is listed the priority of the warning is as listed below not analysed has the highest priority whereas expected has the lowest priority new column Mut Info Here warnings can be present in case
4. are wrong Homof Hetero check Warning H in case homozygous as well as heterozygous exons are present for one gene Splice site check Warning X in case there is a mutation at the splice site first or last two bases of the intron Note This function will only work in case intron sequences are included in the ROI o auto TV If active technical validation is done automatically in case no warnings exist o Reads include PCR primers Please fill out the field choose yes or no in case you have amplicons or PCR primers defined since it improves the assignment to the amplicons auto please change this setting in case you have amplicons defined yes select yes in case the reads include PCR primers no select no in case the reads do not include PCR primers o Mark perfect matches count Reads that have a perfect match to an allele can be marked with a in front of the read in operation Sequence The calculation of the is very time consuming therefore you can enter a read count number into this field By default 2 is entered This means only read that have a count of 2 or higher are marked Reads that have a count of only 1 or not marked even though they have a perfect match to an allele The number can be adapted Note for paired end data all pairs forward and reverse read together that show a perfect match to an allele are marked with a star Here it is possible that the forward read has a match
5. one fastq file per run What s new SEQUENCE Pilot 4 2 0 28 During export a status bar shows which run is exported at the moment In case the mapping to the genome is missing for a run or another error occurs an according message is shown for vcf and bam export 6 12 Operation Sequence 6 12 1 Order Protocol Family Pool On tab Order the name of the Profile used for the Run is shown The new tab Pool is available in operation Sequence In case the file is included in a patient pool the pool name and all other files included are listed In case the file is included in several patient pools several tabs are available on tab Pool 6 12 2 Files ROI Groups Genes Chromosomes and ROls Locations Column Bases is not available anymore The new columns Req Cov and Exp Cov are present now Req Cov refers to all bases that fullfill the settings Required Coverage Settings section in operation Run Exp Cov lists bases whose coverage fullfill the setting Min abs coverage Expected Coverage Warning The percentage of bases with a sufficient coverage compared to the total number of bases is listed The number of bases with a sufficient coverage and the total number of bases are shown in parenthesis Column Coverage was available in section ROl s Location only is now also available in section Files ROI Groups and Genes Chromosomes It lists the following coverage warnings Note Only one coverage warning is listed the priority of the wa
6. panel can be filtered and selected more easily e Press Add X gt to add an ROI for each selected location to the ROI list If for example A E02 What s new SEQUENCE Pilot 4 2 0 43 and A E03 are selected two ROIs are added one for E02 and one for E03 Alternatively press Add 1 2 to add all selected locations as one ROI to the ROI list If for example A E02 and A E03 are selected they are added as one ROI including the intron sequence in between Press Save The ROI List will be empty then because by default not saved ROIs is selected in the field State To see all saved ROIs select the blanc in the field State In the field Suffix special characters can be entered to automatically define the ROI name The default ROI name is GENE LOC suffix o GENE Gene name o LOC Location Exon name Example for gene A E02 o Inthe Suffix field the following entry is listed Test GENE location LOC ende o The following ROI name is created Test A location E02 end Moreover the following columns can be edited in the ROI List Inthe column SeqType the entry is genomic by default This can not be changed e ROls that should only be used for mapping can be defined to filter background reads Therefore the option only mapping can be activated in the field Analysis Mode in the ROI list If only mapping is checked all reads will be aligned to the ROI but it will not be shown in operation Sequence This option i
7. 2 0 5 files that are not affected remains MV an archived order is present and additional result files are loaded for the patient the order is reactivated The same procedure as described for medically validated orders is done e an extracted order is present and additional result files are loaded for the patient the order is reextracted and reactivated The same procedure as described for medically validated orders is done 1 10 Operation Sequence After medical validation button MV the button TV for technical validation is inactive When removing medical validation you can optionally be asked if you are sure to remove the medical validation For this option make the following entry in the 1is ini file in the section of the module CheckMVReset yes This is SeqPilot for modules SeqPatient and SeqHLA SeqNext for module SeqNext SeqNextHLA for module SeqNext HLA e MLPA for module MLPA 1 11 Reports aUAACCCEOCOCOC C LLEA Reports can be saved as pdf files automatically instead of printed when the button Print is pressed If this option is set you can print your Report using Preview Therefore make the following entries in the 1is ini file section Print located in the bin directory of your SEQUENCE Pilot installation A default folder must be defined to save the all Report files ExportDir Reports The file name of the pdf file must be defined as follows The filename can
8. You can display the original view again by selecting the context menu item original sequences in the second combined sequence The split sequence view can also be shown in a separate window Therefore select the context menu item result file view gt split alignment for one of the result file sequences The electropherograms can be shown in a separate window This gives a better overview in case many sequences are present for one location Therefore the new context menu item result file view is available for the result file sequences You have the option to either show the original alignment or the split alignment in the new window Therefore use the combo box original split Edit Bases context menu edit bases in the result file sequence o The context menu in the gene line changed to make editing of frameshift mutations more convenient There is the context menu item 2 allele available A new window opens to select different types By default the type Auto is selected When OK is pressed a frameshift mutations is searched automatically and shown in a new line below the gene line Deletion Here the base sequence of a deletion or the number of deleted bases can be entered The resulting sequence including the mutation is shown in a new line below the gene line after OK is pressed Insertion Here the base sequence of an insertion or the number of inserted bases can be entered The resulting sequence includi
9. also the possibility to install both packages at the same time please keep in mind that in this case both genomes hg19 and hg38 have to be installed The database package can either be downloaded from our homepage or alternatively from our ftp server We recommend to use the ftp server for the download of big files because the download can be restarted in case there is an interruption Download from our homepage Go to http www jsi medisys de genomes snp dbs Download the file hg19 Genome VarDB and or hg38 Genome VarDB After download please verify the integrity of the downloaded file i e whether it is complete and uncorrupted To do so you may use the md5 checksum provided to the right of the respective link Search for md5 checksum on the web to find suitable verification tools Within the verification tool browse and select the downloaded file and let the tool compute the checksum Compare it to the one provided on our homepage If the two checksums differ you have to download the file again Download from our ftp server You can reach the file server using the following link ftp ftpsrv jsi medisys de e Please use the following login Username SeqPilotData Password SeqPilot Download the file hg19 GenomeVarDB exe from folder GenomeVarDB hg19 and or file hg38 Genome VarDB exe from folder GenomeVarDB hg38 The md5 checksums provided with the download links on the website may be used for the respective file o
10. are available 9 5 8 local info jumps to positions with a local info deposited no call jumps to positions that have no coverage expected jumps positions with a coverage below the expected coverage Settings operation Run quality jumps 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 quality in the column Coverage of the ROIs and Locations sections required jumps to positions with a coverage below the required coverage Settings operation Run bg BC jumps to positions with a background warning for base changes If those positions are present there is also the Warning B in the column Warning of the RO s Location table bg Indel jumps to positions with a background warning for indels If those positions are present there is also the Warning N in the column Warning of the ROls Location table next seq for long ROls locations that are not sequenced completely breaks in sequencing moves to the next sequenced part of the ROI Validation There is the option to make medical validation only possible in case a preferred result is set Therefore make the following entry in the 1is ini file located in the bin directory of your SeqPilot installation in the section SeqNextHLA CheckMVPreferredAllele yes 10 Module MLPA 10 1 Operation Joining context menu in the Upper Table o n
11. be created out of DNANo or OrderNo Furthermore a free string can be added to the DNA OrderNo Therefore make the following entries PrintExportFile possible entris are DNANo or OrderNo ExportType PDF ExportSuffix strModule Text e g entry for module MLPA to add the suffix MLPA to the DNA OrderNo ExportSuffixMLPA MLPA 1 12 Jsi Service The processes from the 3siSevice ini file e g SeqResultfiles exe can now be started and ended separately in the Jsi Service window Status What s new SEQUENCE Pilot 4 2 0 6 2 Modules SeqPatient and SeqNext 2 1 Gene Admin In the field Gene you can search for genes by entering letters In case you use splitted genes please set the lis ini entry SeqPilot LoadAlternativeVersion yes It is possible to edit stop codons and to switch off the translation in downloaded gene files Therefore open the gene file located in the folder GeneFiles of your SEQUENCE Pilot installation o Switch off the translation e g for untranslated genes Enter the following into the section of the untranslated transcript s UNTRANSLATED yes o Edit stop codons To overwrite a stop codon make the following entry STOP CODON ExonIndex Pos in the Exon e g STOP_CODON 27 607 This new line has to be entered in the section mRNA Make sure to enter space characters or tabs identical to the other entries in this section 2 2 Operation Seque
12. be shown in the column Warning of the Positions Resultfiles section But the mutation is not used for result calculation alleles are not separated or excluded due to splice site mutations To create an ROI do the following Select the gene in the box Gene Select one or several exons that should be added as ROI the sequence is shown in the field Sequence The field Location shows E 0 0 and the field Seq Range shows 0 and 0 by default only exonic region Here are some examples to explain which sequence is used o E2 0 0 exon 2 o E2 40 0 last 40 bases of intron 1 exon 2 o E2 10 0 exon 2 without the first 10 bases o E2 0 20 exon 2 without the last 20 bases o E2 0 35 exon 2 first 35 bases of intron 3 o E2 20 5 last 20 bases of intron 1 exon 2 first 5 bases of intron 2 o E2 E3 exon 2 and exon 3 including intron 2 in between The sequence range can be changed manually for each location in the field Location or for all locations in the field Seq Range For selected locations an ROI name is proposed in the field Name By default it consists of gene name and exon number e g A E01 This name can be changed Moreover a suffix can be added to the ROI name therefore just enter it in the field Suffix An entry can be made in the field Panel The search field Panel is available in the operations ROI master file ROI Groups master file and Run Therefore all ROIs belonging to one
13. change the link to the GeneFiles folder of your SeqPilot installation Note In case you do not want to create the database folder in your SeqPilot installation but in another directory on your PC you have to enter the path of the directory in the 1is ini file section SeqPilot GenomeVarDir Path e g C GenomeVar GenomeDBVarDir Path e g C GenomeDBVar 4 2 Gene Admin In case a gene file with an entry in Gene Admin can not be found in the GeneFiles folder of your installation no txt file available or txt file is corrupted and the genome hg79 is installed hg19 sequences are present in Gene Admin With these no SeqPrimers and AmpModules should be defined since all transcripts are active for hg19 The first transcript listed in the soforms table is always used 4 3 Operation Joining The context menu item settings Upper table is available for several selected files The settings Gene Amp Module Seq Primer and DNA No can be changed for all selected files simultaneously 4 4 Operation Sequence 4 4 1 Electropherogram Below the location overview a peak height ratio diagram for each base position is available The bars show the background for each position In case there are no or only small bars above below the middle line there is no high background In case a background is present there is a blue bar above the line for forward bases and a purple bar below the line for reverse bases The bars are c
14. context menu All ROIs included in the file can be exported as BAM or FASTQ files Therefore use the context menu item export For both exports the following is done A directory to save the data can be selected Alternatively you can create a default directory that always should be used To use this option enter the path of the export folder in the 1is ini file section SeqPilot behind the entry ExportDir When the export is finished the number of generated files and the path of the export folder is shown The file name is created out of the DNA number Note The export also works for ROIs Locations context menu in the section RO s Location and in operation Joining context menu in the Lower Table for one or several selected Runs reads BAM creates one BAM file and one BAM BAT file for the complete file The following additional information is exported as a comment in the bam file SeqPilot version CO BAM file generated by SeqPilot path and name of the imported raw data file s CO InputFiles Settings CO Profileld Each reads gets a tag behind the quality with the profile name of the ROI the read was assigned to e g PF Z CNV PF stands for Profile a Z date CNV is the profile name PM additional tag profile modification can be present in case the profile for one ROI was changed Note Reads that are aligned to several ROIs can be present in the bam file sev
15. covered by reads show fragment size Only present for sheared paired end data without amplicons e g Haloplex data The setting randomly sheared has to be set in the Settingsloperation Run For these data the fragment size is checked This gives a hint for deletions insertions Too short fragments give a hint for an insertion too long fragments a hint for a deletion When the item is used the fragment size is shown graphically below the location overview Moreover the following values are listed Avg Average fragment size for the location Standart deviation Max Maximum fragment size in the file Min Minimum fragment size in the file In case a peak is present that exceeds the doubled standard deviation there is also a Warning in the Variation Mutation table tab Warning The warning is defined in column Hint there is the entry fragment size possible deletion for a deletion and fragment size possible insertion for an insertion Reads view check box original reads There are differences in the reads view depending on if the normal view combo box combined or fwd rev or extended view combo box combined ext or fwd rev ext is selected in the electropherogram o normal view Before after the ROI a maximum number of seven bases is highlighted Bases are not shown o extended view The reads sequences in the extended area are shown automatically in case they can be aligned to the gene reference sequence After the ex
16. e The new condition F is available in the column Condition This condition is present in case the new frameshift analysis algorithm was used automatically Note in case a Resultfile is set to ignore automatically and the Condition F is present the exon might have been ignored due to a frameshift The following new context menus are available o editing gt frame shift analysis on off using this the new frame shift analysis algorithm can be switched on or off o export gt 2 allele sequence The sequence of the second allele can be exported as a seq file The second allele is the non wildtype allele This is helpful to copy sequences with frameshift mutations o copy gt 2 allele sequence The sequence of the second allele can be copied o fequirenment gt cancel To cancel requirements all open requirements can be selected simultaneously now The second allele is the non wildtype allele This is helpful to copy sequences with frameshift mutations 4 4 4 Variation Mutation table The table can either be shown for the gene selected in section Genes or for all analysed genes Therefore use the radio button all Genes selected Gene above the Variation Mutation table The new mutation type nde is available This defines several mutations lying very close to each other This can either be two several base changes or base change and deletion insertion By default the maximum number of bases between two base chan
17. file can be done In this case the dialogue Change Gene Transcript is empty The new button Copy is available a copy of the selected RO Group is saved You can enter a new ROI Group name and lot number The following new search fields are available o Suffix here you can search ROIs with a certain Suffix Therefore press Search behind Suffix o Source Here you can search for ROIs with a certain Source The following sources are possible gene file genome chromosome range and fasta file The following new table columns are available e Index the table entries are numbered o Source Here the source of the sequence is defined The following sources are possible gene file genome chr range and fasta file o Analysis Mode in case the entry is only mapping the ROI is not shown in operation Sequence can be set in operation ROI master file o Settings Profile special Profiles that are used for analysis of the ROI are listed can be set in operation ROI master file The context menu entry show ROI info is available in the RO Group table to open the ROI info window Button Import Primer The following is checked when a list of primer pairs is imported o 1 case no amplicons are present all primer pairs that can be mapped to the extended ROI are added as amplicon inlcuding primer sequences o 2 case amplicons are present already for the ROI but without primer sequences The primer sequences are o
18. is listed in paranthesis behind the ID 2 entry Overview Positions with WebRefs DB entry are shown in the location overview and electropherogram highlighted grey Information about the WebRef is shown in a tooltip when moving over the gene reference sequence Moreover information for variations mutations present in the SNP databases can be shown in additional columns of the Variation Mutation table Please have a look at our User Manual SeqNext for detailed instructions The following items on tab Settings changed o The Profile Settings selected in operation Run are only applied to the ROIs without profile settings assigned to them column Settings Profile is blanc in operation ROI master file o The name of the used Profile is available in operation Sequence tab Orders and on the Report o The tab Hide Mutations is not available any more The options that were present are available on tab FiltersAab JSI o The following settings changed tab Settings e section 2 Analyse ignore region was renamed into Required Coverage section 3 Coverage Warning was renamed into Expected Coverage Warning The Low abs coverage warning was renamed into Min abs coverage e Randomly sheared this setting can be activated in two cases o Reads are amplicon based amplicons defined and sheared afterwards This setting is obligatory in case amplicons are defined Primers are removed e Randomly sheared DNA no amplicons defined i
19. master file 6 6 1 All tabs New column Restrict in the ROI List Note the column Restrict is inactive by default Therefore the ROIs also old ROIs can be shown in the extended view in operation Sequence for new Runs There are three differences present between restricted and extended not restricted ROIs 1 Not restricted ROIs can be shown in the extended view in operation Sequence ROIs are shown in an extended view when the combo box combined Sequences Electropherogram is switched to combined ext or fwd rev ext The ROI is extended at the 5 and 3 end The extension is as long as the longest reads that overlaps with the ROI Moreover the Coverage in the extended area must reach the Required coverage Min abs coverage set in the Settings in operation Run 2 In the extended area no mutation calling is done Exception are indels dels that partly lie within the ROI but reach over the ROI beginning end see chapter 6 12 7 1 for detailed information for extended ROIs no amplicons defined the complete mutation is called also the part that lies outside the ROI for restricted ROIs the deletion indel is not called completely Only the part that lies within the ROI is called In this case there is a sign bin front of the deletion No amino acid change is calculated 3 The copy number of the reads number in front of each read in operation Sequence and the Reads view is also influenced Identical reads are liste
20. o In the field M Ds Barcode the MID list is available This requires that the MID list is stored in the SEQUENCE Pilot installation see User Manual o The new tabs Filters and Show Var Dbs are available to edit these settings for the Run New context menu items in the Lower Table for one ore several selected orders expand collapse order Here all orders or the selected order can be expanded or collapsed to show or hide the joined files respectively e export gt also works for several selected runs For all exports the following is done A directory to save the data can be selected Alternatively you can create a default directory that always should be used To use this option enter the path of the export folder in the lis ini file section SeqPilot behind the entry ExportDir When the export is finished the number of generated files and the path of the export folder is shown The file name is created using the DNA number o variations vcf file The variations of all selected run are exported as vcf file one vcf file per run o reads bam file exports the reads of all selected runs as bam file one bam file per run This file contains already trimmed but not aligned reads Moreover the used Profile software version and the Input file link is exported as well The bam file can also be exported in operation Sequence context menu in section Files o reads fastq exports the reads of all selected runs as fastq file
21. operation Run The according mutations are also marked pink in the Variation Mutation table Filter Step In case mutations were filtered filter DB or settings the filter step where the mutation was filtered is listed 6 12 8 Electropherogram Sequences In the tooltip of forward reverse and combined sequence the average quality score value for each base position is shown in brackets behind the base ROls can be shown in an extended view Therefore switch the combo box combined in the Electropherogram Sequences part to combined ext In case you use the fwdtrev setting select fwd rev ext The ROI is extended at the 5 and 3 end the extension is as long as the longest reads that were aligned The copy number of the longest reads have to exceed the setting Min abs coverage In the extended area the following sequences are shown o n case amplicons are defined in the extended area no sequences and coverages are shown except for the gene reference sequence o n case no amplicons are defined the pseudo electropherogram coverage graph forward and reverse sequence are additionally shown sequences are only shown in case they can be aligned to the reference Variations Mutations are shown but not called Reads sequences are not displayed Note o The reads sequences in the extended area can be shown in the Reads view check the box org Reads Here only reads bases that can be aligned to the reference are shown What s new S
22. or no sign equal in front in the hierarchy genomic DNA cDNA of total What s new SEQUENCE Pilot 4 2 0 9 Examples o gt 6 search for mutations found in genomic DNA in more than 5 orders o 10 search for mutations found in cDNA in 10 orders o 25 26 search for mutations found in genomic DNA in more than 5 orders and in cDNA in more than 6 orders o of 20 search for mutations found in less than 20 orders CDNA and or genomic DNA Disease No Lists mutations with a special disease number only The disease number is shown in the dialogue Mutation Ethnicity Lists mutations with a special Ethnicity only The Ethnicity is shown in the dialogue Mutation Organ Lists mutations with a special Organ only The Organ is shown in the dialogue Mutation Phenotype Lists mutations with a special Phenotype only The Phenotype is shown in the dialogue Mutation 2 3 4 Other new features Big insertions deletions gt 30 bp can be added to the mutation database All sequence information is available now in the fields Nuc Change Nuc Name AA Change and AA Name You can search for a gene by entering letters using the search field Gene The following new items are available in the context menu of the Gene table for one or several selected gene s o delete mutations without orders Mutations with no Orders listed in section Orders e g imported mutations are deleted o set transcript a transcript can be set see chapter
23. possible mutations are listed on tab Warning of the Variation Mutation table cutoff Indel Fragment size orientation Tab Gene RO new column CNV that lists distinct mutations detected by CNV analysis Tab ROl Amplicon The average coverages fwd rev on tab ROI and tab Amplicon are listed in two separate columns now Average quality scores for fwd rev are listed in the column QS fwd avg and QS rev avg on the tabs ROI and Amplicon Information about coverage warnings is displayed percentage of bases with coverage warning number of bases with coverage warning total number of bases The following columns are present o No call Positions with coverage warning no call o Required Positions with coverage warning required o Quality Positions with coverage warning quality o Expected Positions with coverage warning expected New column Called bp shows the number of called bases percentage of called bases number of called bases number of ROl amplicon bases New column Mean RD shows the mean read depth The columns Multiplex No and Comment can be shown Therefore open the context menu Mangage table columns click on the header and increase the Width for the corresponding columns Tab Amplicon New column Dupl Reads shows entries in case SmMIP Processing is used only see chapter 6 3 New columns Multiplex No and Comment show the Multiplex number and Comment respectively that was entered in operation ROI
24. present in the folder Settings SeqNext of your installation In the MID txt file the MIDs have to be entered in the following way MID 1 ACGAGTGCGT MID 2 ACGCTCGACA MID 3 AGACGCACTC MI D 4 AGCACTGTAG For the Autorun txt file three new fields 9 10 and 11 are available The fields have to be entered in the following order 1 2 3 ao p 10 11 DNA No Barcode optional or MID MID is only possible if the MID is present in the MID list ROI Enter the name of the ROI s and or ROI Group s an ROI Group with a Lot has to be entered as follows ROI Group Name Lot There has to be a blank before and after the slash Path of the file s Organ Phenotype optional has to be entered in the following way Organ Phenotype Type optional in case no type is entered the type is patient automatically Settings optional in case no settings are available the default settings saved in the lis ini file are used If not the default settings are used the settings have to be saved in profiles If you want to use a saved profile give the profile s name as settings entry e g MyProfile There must not be a FieldSeparator default in the profile s name or before or after it Project optional Organism optional Filter optional enter a Filter profile If nothing is entered the default profile is used ShowVarDBs optional enter a ShowVarDBs profile If nothing is entered the default pro
25. sections Files ROI Groups Genes Chromosomes and ROls Locations e SeqNext HLA Warnings Warnings that occur in column Warnings in sections Files ROI Groups Genes Chromosomes and ROls Locations e SeqNext UnusedReadsErrorCodes Explains possible error codes for unused reads 1 6 Operation Users master file For Client Server installation the new User Right edit scheduler is available If active the Task Scheduler Menu System item Task Scheduler can be used 1 7 Operation Orderlist Extract archived orders When the button Extract is pressed you can decide if you want to extract all orders or the selected orders by pressing all or select n the search field Module the new option undefined module is available 1 8 Operation Projects master file A search function is available for projects You can search for projects with a certain Name Comment or State active inactive 1 9 Operation Joining The new lis ini entry JoinRFValidatedOrder yes is available for all modules now For module SeqPatient and SeqHLA the entry has to be set in section SegPilot for all other modules in the section of the module e g for module SeqNext use the section SeqNext The following cases can be present a medically validated order is present and additional result files are loaded for the patient the result files are joined to the order and the order state is reset to complete The state of result What s new SEQUENCE Pilot 4
26. the column Files The coverage warnings expected no call drop out required quality for an ROI can be shown on the Report Therefore please contact our support team The mean read depth Mean RD can be printed on the Report Therefore please contact our support team 6 13 Operation Archiving Archiving makes the result data smaller In the SeqNResults folder only the file ROT name txt e g BRCA1 E02 txt remains All other files such as Frags txt and UnusedReads txt are deleted Therefore after archiving all data can be viewed but no recalculate edit can be done any more The following data can not be viewed anymore UnusedReads section Files or ROls Location context menu show unused reads Summary on tab RO and Amplicon not all information is present anymore These are columns Called bp Required Quality and Expected Assigned Reads and Aligned Reads on tab ROI and all columns on tab Amplicon 6 14 CNV analysis 6 14 1 Operation ROI master file Multiplicom MASTR assays including controls for CNV analysis are now imported on tab Add Panel The following fields have to be changed in the window mport Panel file o CNV Control 4 o Key Control o Plex No this is not present in the file yet Moreover in the section Settings activate the options build amplicons The new column CNV probe type lists the CNV probe type For control ROIs this entry can be set to Control blanc means Target R
27. the downloaded Pseudogene org file can locate in any folder on your PC To create an ROI Group containing pseudogenes from Pseudogene org Enter a Group name in the field Name and press Save Press the button Import Pseudogene org file A new dialogue opens Here you can select o The Group Name o Optionally a Lot Number o An ROI Name Prefix In case a Prefix is added in front of the ROI name o Organism Select the genome that should be used for mapping of the pseudogenes Select the downloaded file Pseudogene org file Please note that the import takes several minutes Pseudogene ROIs are created automatically The Pseudogene ROIs are named ROI Name Prefix Pseudogene org identifier the Panel is Pseudogenes org They are set to Analysis Mode only mapping therefore pseudogene reads are filtered and not shown as ROls in operation Sequence Note In case the pseudogenes can be mapped to the default genome the chromosomal location is shown the SeqType is genomic Sequences that can not be mapped are stored as fasta sequences They are also used for filtering For pseudogene filtering the ROI Group has to be selected in operation Run in addition to the real ROI Group All reads that can be mapped to known pseudogenes are filtered Filtered reads are listed in the file UnusedReads txt that locates in the SeqNResults folder of your SeqPilot installation in the folder for the order The reads have the fl
28. the quality filters The mapping and alignment information is used from the bam file tab Expert Settings Min Homozygous the percentage coverage a mutation has to reach to be called homozygous instead of heterozygous can be set In the previous version the minimum coverage for homozygous calling was 100 Ignore coverage automatically Allow unique paired end reads By default this setting is not active In case a read pair does not align to the same ROl amplicon both reads are discarded If this setting is active the unique reads are aligned to different ROls What s new SEQUENCE Pilot 4 2 0 26 6 9 2 Patient table For one patient per gene analyses it is now possible to enter several patients without barcode in the section Patient In this case different ROIs have to be defined for each patient Instead of joining the reads to a patient by MID the reads are then joined to a patient by ROI Import of a patient list Button mport Instead of a barcode an MID number can be imported Note In this case the MID list MIDs txt has to be present in the folder Settings SeqNext of your installation In the MID txt file the MIDs have to be entered in the following way MID 1 ACGAGTGCGT MID 2 ACGCTCGACA MID 3 AGACGCACTC MID 4 AGCACTGTAG 6 9 3 Autorun Instead of a barcode an MID can be entered into the corresponding field of the Autorun txt file Note In this case the MID list MIDs txt has to be
29. withing the ROI a 2bp deletion is called In the Variation Mutation table there is a in front of the deletion No amino acid change is calculated no entry in column AA change 6 12 7 2 Tabs The following new tabs are available filter lists mutations that were filtered permanently using the SNP database filter operation Run operation Joining operation Sequence ROI table temp filter lists mutations that were filtered temporarily using the SNP database filter or the settings filter in the Variation Mutation table context menu filter DB or settings respectively The tab is cleared when the order is left warning This tab lists entries of the Type W These are warnings that give a hint for possible mutations By default the entries listed on this tab are not listed on tab all You can change this using the context menu in the Variation Mutation table settings gt add warnings to tab all To remove warnings from tab all use setting gt remove warnings from tab all In case entries are present there is also an corresponding entry in the field Mutation in sections Files Genes ROI Groups and ROls Locations In case there are mutations called in an area with a Warning these mutations are marked with a W in the Location overview The warnings are further defined in column Hint the following entries are possible o cutoff left right This warning is shown in case reads are present where a part fits perfectl
30. 2 3 1 o set genomic cDNA the SeqType genomic cDNA can be set see chapter 2 3 2 The following new items are available in the context menu of the Mutation table for one or several selected mutation s o set transcript a transcript can be set see chapter 2 3 1 o set genomic cDNA the SeqType genomic cDNA can be set see chapter 2 3 2 The new columns Changed Date and User are available in the Mutation table o Changed Date date when the mutation was changed last in the dialogue mutation o User User ID of the user who added or changed the mutation Dialogue Mutation context menu show mutation o The fields NucName and AAName are in read only mode They can be changed using the button Set behind the corresponding field o Button Reset Frequency When this button is pressed you are asked if you are sure that you want to reset the frequency If Yes is pressed the frequency is reset Orders table new column Module is available which lists the module that detected the mutation SeqNext and or SeqPatient The information is only present for newly added orders not for existing orders In the Mutation dialogue as well as in the Orders table the sorting of orders begins with the What s new SEQUENCE Pilot 4 2 0 10 latest order Several genes can be selected simultaneously for export button Export 3 Modules SeqPatient and SeqHLA 3 1 Operation Joining The search for orders is quicker HLA database genefil
31. Amplified Geneparts A new table is available that lists all exons of the gene selected in the field Gene Moreover the exon length is shown in the column Exon length and the columns Prev Intron length and Next intron length show the number of intron bases flanking each exon In the table one or several exons can be selected The selected exon s are listed below and set automatically New field SeqType here you can select if you analyse genomic or cDNA For cDNA no introns are included 8 3 Operation SeqPrimer master file The following fields changed SeqPrimer gene parts A new table is available that lists all exons of the gene selected in the field Gene The table lists all exons of the gene selected in the field Gene Moreover the exon length is shown in the column Exon length and the columns Prev Intron length and Next Intron length show the number of intron bases flanking each exon The field below shows 0 0 by default This defines the number of intron bases that should be included in the SeqPrimer on each side of the exon With the default setting no intron bases are included Gene parts are defined as follows o E1 or E1 0 0 the complete exon 1 is amplified o E2 20 0 exon 2 is amplified without the first 20 bases o E2 20 20 exon 2 with the last 20 bases of intron 1 and the first 20 bases of intron 2 o E2 0 20 exon 2 without the last 20 bases o E1 E2 E3 or ET1 0 E2 E3 0 exon 2 to 4 are amplified
32. EQUENCE Pilot 4 2 0 36 Parts that can not be aligned are shown as grey bars The complete read sequence can be shown with the context menu item show origninal reads for a selected read o The extended view is only available in case the ROIs are not restricted To switch off the extended view function completely for one or several ROIs activate the column Restrict in the operation RO master file In case you activate the column Restrict deletions indels that partly lie within the ROl amplicon but reach over the ROl amplicon beginning end are not called completely Only the part that lies within the ROl amplicon is called Identical reads In the old version identical reads were listed only once There copy number is shown at the left end of each read This changed Now reads are only identical in case the complete alignment is identical To see the complete alignment open the Reads view and check the box org Reads Here all bases must be identical also bases that are present in the grey areas outside the ROl amplicons e Location overview o The following additional information is shown in the tooltip in the Location overview Genome Chromosome TranscriptID average coverage and average quality for each amplicons and Warnings o There is a context menu available Show read coverage shows the read coverage not the coverage for each position For deletions the drop in coverage is not shown anymore in case the deletion is
33. Next for further details The following Settings changed Map to gene files Gene Admin activate this options in case gene files from Gene Admin should be used to set up the panel In case a gene is not present in Gene Admin the gene from the genome selected in the field Organism is used Cut Expand is inactive by default field is empty The ROI sequences can be cut expanded to the values set in the fields 5 and 3 The entries in field Location change The following options can be selected o cut exons Exons are cut to the values entered in the field 5 and 3 for each ROI e g Setting is 5 20 and 3 30 Exon is cut to 20 bases before and 30 bases after the exon Note ROIs are only cut and not expanded therefore this setting can be used for amplicon based sequencing o Cut expand exons Exons are cut or expanded to the values entered in the field 5 and 3 for each ROI o cut coding exons refers to the coding sequence from start to stop codon e g setting is 5 20 and 3 30 all ROIs are cut to 20 bases before and 30 bases after the exon In case the ROI includes a start codon stop codon it is cut to 20 bases before the start codon and 30 bases after the stop codon In case non coding exons are present they are set up as ROIs but set to only mapping in column Analysis Mode Note ROIs are only cut and not expanded therefore this setting can be used for amplicon based
34. OI In case a panel file is imported Add Panel file and the control ROls have an common What s new SEQUENCE Pilot 4 2 0 38 identifier this information can be entered in the mport Panel file dialogue in the section CNV controls enter the column number into the field CNV control and the identifier in the field Key If the identifier is present for an ROI the CNV probe type is set to control automatically The Plex number for a Multiplex PCR can be saved now Therefore the Plex numbers can be added in the section Amplicons PCR Primers column Multiplex No In case a panel file is imported and the plex number is present in the file the column number can be entered in the field Plex No of the Import Panel file dialogue for automatic import 6 14 2 Operation Analysis mode CNV master file The new field Multiplex No is available in the Group ROIs list Entries into this field can be done in operation RO master file It can be used for sorting the ROIs and easy definition of the analysis modes in case several Multiplex PCRs were done The controls that are joined to patients automatically can not be set in operation RO Groups master files any more This option is now availabe in operation Analysis mode CNV master file The section CNV Group has the following new options o activate the check box TV controls only to join only controls that are technically validated o new control setting Autorun RunID This option is useful wh
35. Press Search In the result table it is now listed in which genes ROIs the sequence was found Moreover the number of mapped aligned fwd rev is shown in the corresponding columns unused reads A new window opens that lists the reads that were unused not mapped or aligned to an ROI The same information is also present in the UnusedReads txt file In the section Overall all errors that occurred are explained e g Error 51 reads have a quality score not satisfying the user specified threshold The number of reads filtered for each error mapping alignment is listed You can find a list of all possible errors by pressig the button in this window or in the Menu Helplitem UnusedReadsErrorCodes In the middle a search for unused reads of certain Genes ROIs Error codes or Sequences can be done If the button Search is pressed o the section Searched lists the information about unused reads depending on the search What s new SEQUENCE Pilot 4 2 0 30 criteria o the table below displays all reads that fullfill the search criteria The following information is given ROI name of the ROI or Homology region the read could be mapped to If the read could not be mapped to an ROI this field stays empty Error Error code number Base seq Base sequence of the read One or several selected base sequences can be copied Therefore do a right click on the sequence and select copy selected base sequence s from the
36. Primer The following is checked when a list of primer pairs is imported 1 1 case no amplicons are present all primer pairs that can be mapped to the extended ROI are added as amplicon inlcuding primer sequences 2 2 case amplicons are present already for the ROI but without primer sequences The primer sequences are only saved in case they enclose an existing amplicon Otherwise the primers are not imported you will get a list of invalid primers and possible ROIs No new additional amplicons are added 6 6 2 Filters for background reads There are two new options available to filter background reads e g pseudogene sequences in operation ROI master file Button Find Homologies An automatic search for homologous regions in the genome can be done for ROIs to exclude background reads Therefore select one or several ROIs in the ROI List and press Find Homologies The genome is searched for homologous regions for the selected ROls In case they are present the chromosomal positions are entered in the new section Homologies only mapping for each ROI Reads that are mapped to the ROI and match to active homologous sequences are discarded Note When this button is used for the first time bwa files are created in the Genome hg19 What s new SEQUENCE Pilot 4 2 0 19 folder of your SeqPilot installation default C SeqPilot GeneFiles Genome hgl19 This first time creation takes around 1 2 hours To skip this proce
37. What s new SEQU ENCE Pilot Version 4 2 0 02 05 2015 SEQUENCE JSI Pilot medical systems GmbH www jelmedisys com 5 gis I gi ge genet developed by JSI medical systems GmbH Tullastr 18 77975 Ettenheim GERMANY phone 49 7822 440150 0 Fax 49 7822 440150 20 email mail jsi medisys com web JSI medical systems Corp 1215 W Imperial Hwy Suite 205 Brea CA 92821 USA phone 1 714 332 0139 fax 1 714 332 0131 email mail us jsi medisys com www jsi medisys com for research use only Table of Contents URS Rc c9 err cc MEME HERR REOR E E 4 1 1 64 bit database cc ccccccccccccccceeeeeeeeeeeeeceeeeaaaaeeeesaaaeeeeeeeeeeeeeeeseeeaaaaaaaaeesaaeeeeseeeeeeeeeeseeseanaaaaaees 4 1 2 Automatic TIMEOUN TT 4 296 4 ES 0 Es e E E 4 EN CUN PN e RE 5 1 5 Operation Users master Tile reed veo tarea C REEL ERREUR ER FERE RU Er EHE DIE EFE rd 5 Tur Operam ENE aspis prop ete AA PETE PEN EE EGUT ae aise aa 5 1 8 Operation Projects master file ssseesssssssseeeeeeeeeeenennn mmm 5 LESER OD PP METRI PP ee PE 5 1 10 Spero SSW CS ii iwcdatiavientraridasemareaGdinmigninadussonaibendieGaeitidlanaineacenunt 6 3 13 REPONS coinsias ra E eE a S 6 UE cud ec eT a E A E E A E E EN T MER 6 2 Modules SegPatent and ri oM Emm 7 AMISSIS nM X 7 2 2 pera
38. Y ad Ee REM FREREDER EPUM E OSE AU EI px EE 15 5 1 PAROISSE ING Sr PO SUNT uuo air HA ERE AE n ens aema E Pea iUd n Cose E osRT DO 15 9 2 Operation Users master TO eec a E EAEE 15 SE Ea E E E RN E E E A E E E a TT E E A E 15 o Moie So Ne Kennissen AAA a a M 15 SOME GUT MRNA DU DNE DP EEE 15 6 2 import or Yariation DD files ice iier persa a uade a n e nb nerdezenabos RE A A Ra CEn E RM A RAN OS E REE 15 Do File Unused Reais DE aosoiadas tern uk d ena ee dre a eae 17 SE E uum NR I a eiri 17 pS Menu PIOI sonini r e 18 6 6 Operation ROI master WO uiis iier a Per Pn Oei a DC 9 Pre E ERE HOO Fa Hd aie 18 DB AU Paes acids TORO EDOERELEEDEREEEEVRLHEUREEE DERE DEREN DEDE DEVE ed ea ET 18 6 6 2 Filters for background Dico MEN REN D TE 19 6 6 3 Tab add PCR and tab add Panel eesssssssssssssssseeeneneee rere ener nnne 20 60 4 Tab add PORNAD ODOlIB abe ro RER A RCHRH HALE b E EEEE 20 6 6 5 Tab add PORNAb F asado rd ROREROVEE ELI RR AES FRU OLD EC HAN EA UR EE t E rM Hz Ea El ES 21 DESEINP a Loch i as cse pads diveniceci chnceetatonsencedececzads 21 6 7 Set up or Mulliplicor MAS TR asSayS dii ivive ak ep DEAE ER HEEL E HR E E FER PEE Ed 22 6 5 Operation ROI Groups master TG usxexciexikt ixi v Had upp FERR ER ERE Kad rt o FO DEDI HER HEURE CF ri 23 6 8 1 Import of Pseudogenes from Pseudogenes org eese 24 oa Opara on IS DEE soia eo oreet uou tui Dd c acis mi ERR LI uU Pct inen mE cu uds i uM 25 09 1 i as i
39. ag 4 and the name of the Pseudogene in front Filtered reads can also be viewed with the following context menu in operation Sequence sectionFiles show usused reads What s new SEQUENCE Pilot 4 2 0 24 6 9 Operation Run 6 9 1 Section Settings New tab Filters here special Filters can be defined For filtering special internal filters JSI can be used as as well as imported databases ClinVitae COSMIC ClinVar 1000 Genomes dbSNP Mutations that do not pass the filter settings are listed on tab Filter in the Variation Mutation table A filter profile can be saved Please have a look at our User Manual SeqNext for detailed instructions New tab Show Var Dbs Information for mutations variations present in the gene files Gene Admin and in the SNP databases can be shown in the Variation Mutation table and sequences A profile can be saved One tab is present for the gene file in case a gene file from Gene Admin is used as reference sequence and for each imported SNP database The number of variations mutations described in the databases is listed on each tab as well The Profile default is present already The first two entries Variation and Overview are selected in column Show on all tabs Genefile Gene Admin and SNP databases Therefore the following is displayed by default 1 entry Variation Reference IDs are shown in the Variation Mutation table column web Ref for detected mutations The database
40. alculated as follows peak height ratio peak area highest not reference bases peak area highest not reference base peak area reference base There is a context menu available in the peak height ratio diagram to show the length of the result files below the location overview o show peak height ratio default setting the peak height ratio diagram is shown below the location overview o Show result files the length of the result files is shown below the location overview What s new SEQUENCE Pilot 4 2 0 12 Reverse sequences are highlighted in a darker color o show original result files the length of the original result files not shortened due to quality SeqPrimers are shown Sequence parts that are not used for analysis are highlighted grey The detection of frameshift mutations was improved especially for more complex cases It is now possible to detect SNPs after before a frameshift The new mutation type ndel is therefore available The new context menu item split sequences is available in the combined sequence Using this the combined sequence is split into two homozygous sequences Moreover each result file sequence is split into two homozygous sequences The two homozygous sequences are virtual sequences representing the expected sequences if the two alleles would have been sequenced separately This gives a better overview to resolve more complex sequences In this view only mutated positions are marked red
41. analysis is completed the file can be joined to the order again using the button Autojoin The dialogue nfo can be opened with the context menu entry show Info in the Upper and Lower table was adapted 9 5 Operation Sequence 9 5 1 Files Genes ROIs and Locations All sections Several warnings can be present in the column Warning If a warning is shown depends on your settings in the operation Run Note In case the Warnings are present in the intron sequences they are only shown in sections that show intron data These are Files ROIs and the first entry of Locations What s new SEQUENCE Pilot 4 2 0 49 F warning Min reads per allele number of reads joined to allele 1 or allele 2 is below the corresponding setting A Warning Allele 1 Allele 2 Proportion ratio of allele1 to allele 2 is below the corresponding setting B Warning Basecalling 6 coverage background a third allele exceeds the settings Use the jumper bg BC in the dialogue Show to jump to the corresponding positions N Warning Basecalling Indel background Warning N in case the coverage of an insertion deletion exceeds the settings Use the jumper bg ndel BC in the dialogue Show to jump to the corresponding positions P DRB pseudogene plausibility check DRB1 expected pseudogenes are wrong H Homo Hetero check Homozygous as well as heterozygous exons are present for one gene X This warning is shown when a mutation is present at the first or l
42. ases and a bar below the line for reverse bases The higher the bar is the bigger is the background For typical heterozygous positions bars are present for forward and reverse bases The bars are calculated as follows peak height ratio peak area highest not reference bases peak area highest not reference base peak area reference base o show result files default setting the length of the result files is shown below the location overview Reverse sequences are highlighted in a darker color o show original result files the length of the original result files not shortened due to quality SeqPrimers are shown Sequence parts that are not used for analysis are highlighted grey New section Groups Here an entry is available in case SeqPrimer groups exist for the used SeqPrimers When the entry is selected the electropherogram and sequence data is shown for all grouped SeqPrimers as one sequence The length of the resultfiles are indicated as bars in the location overview The new warning X is available in the Positions Resultfiles tablelcolumn Warning This warning shows that there is a mutation present at the first or last two bases of the intron splice site Note This function only works in case intronic regions are defined in operation SeqPrimers master file Section Show the new field show is available Here you can select o Exon it is only jumped to exon positions when the jumpers in section Show are used c
43. ast two bases in the intron Note This function only works in case intronic regions are included in the ROI Sections ROIs and Locations Section ROIs lists all defined ROIs used for the Run Section Locations lists all exons that are covered by the selected ROI For the location selected here sequences are shown in the electropherogram Introns are only displayed if the first entry ROI is selected Several Coverage warnings can be present in the column Coverage of section RO s and Locations Note Only one coverage warning is listed the priority of the warning is as listed below not analysed has the highest priority whereas expected has the lowest priority not analysed an error occurred Please recalculate your file dropout No sequences are available for more than 9096 of the ROI No sequences are also present in case the Required Coverage Settings Min abs coverage and or Ratio read directions defined in operation Run section 2 are not fullfilled nocall There is no coverage at one or more positions of the ROI Only occurs in case no Required Coverage Settings are set required This warning shows that positions in the file are ignored not analysed Positions are ignored in case the Settings for the Required Coverage Min abs coverage and or Ratio read directions defined in operation Run section 2 are not fulfilled The ignored positions are greyed out in the sequences forward reverse and combined sequence
44. column Mutation Here warnings can be present in case possible mutations are listed on tab Warning of the Variation Mutation table The following warnings can be present o cutoff Reads are present where a part fits perfectly to the reference and starting from a certain position the bases can not be aligned anymore e g as expected for transversion translocations A warning is only shown in case the frequency of the sequences that can not be aligned reaches the Warning value defined in the Settings operation Run Expert Setting Warning default 5096 Hint in the Variation Mutation table is cutoff left right What s new SEQUENCE Pilot 4 2 0 29 o Indel There are possible deletions insertions in the ROI that were not called The warning is present in case there are different overlapping insertion deletions present for at least one position of the ROI Together the coverage of these insertions deletions at a position must fullfill the Settings Profile selected in the operation Run Hint in the Variation Mutation table is possilbe del possible ins fragment size Only present for sheared paired end data without amplicons e g Haloplex data The setting randomly sheared has to be set in the Settings operation Run Deviation from standard library size might hint at a large insertion or deletion Too short fragments give a hint for an insertion too long fragments a hint for a deletion In the Variation Mutation table the Hint fragment size p
45. ctory Genefiles Fasta of your SEQUENCE Pilot installation by default this is C SeqPilot Genefiles Fasta This was changed because for Client Server installations no absolute path for the location of the asta file was allowed 6 6 6 Tab Add Panel The tab add Enrichment Kit was renamed into tab add Panel New option to save a panel during set up During creation of the ROls the entries of the table on the left side can be saved This allows to interrupt the ROI set up e g selection of the correct transcript before the ROIs are added to the ROI List To save the table on the left side enter a name into the field Panel Then press Save behind the field To edit a saved panel press Change behind the field Panel After the set up is complete ROIs are added to the RO List and saved the saved Panel on the left side can be deleted Therefore select the corresponding entry in the field Panel Name and press Delete behind the field Window Import Panel File After Build is pressed the window mport panel files opens automatically This window also can be opened by pressing Columns in the section Settings Entries in the window mport panel file changed Note the entries in this window are filled out automatically depending on the used panel file You can edit columns containing special information such as gene name transcript ID and information about controls for CNV analysis Please have a look at the User Manual Seq
46. d only once the copy number is listed in front of the reads Identical means the following for restricted ROIs the reads must be identical within the ROI or amplicon borders for extended ROIs the complete read sequence that can be aligned to the extended ROI must be identical This is the sequence that is shown in the Reads view when org Reads is active For this reason for extended ROIs reads can have a lower copy number as for restricted ROIs The column Restrict can be changed for all or selected ROIs simultaneously by using the What s new SEQUENCE Pilot 4 2 0 18 context menu in the ROJ list The new entry Restrict gt set remove gt all selected is therefore available Settings can be selected for each ROI In the new column Settings Profile an existing Profile that should be used for this ROI can be selected Alternatively the item settings can be selected in the context menu for the ROIs When a Profile is created or selected the selected profile is set in the column Settings Profile automatically The selected Settings are always used for the analysis of the ROI even if another setting is selected in operation Run A profile can be entered for several ROIs simultaneously Therefore select several ROIs in the ROI List and use the context menu item settings to set a profile The following new search fields are available 1 Suffix here you can search ROls with a certain Suffix Therefore press Search behind S
47. e in the dialogue Mutation What s new SEQUENCE Pilot 4 2 0 7 To set transcripts you have the following options You can set transcripts for all mutations of one or several gene s Therefore select one or several gene s in the Gene table open the context menu and select set transcript You can set a transcript for one or several mutations Therefore select the mutation s in the Mutation table open the context menu and select set transcript For both options the new dialogue Transcript opens n the field Operation you can choose to automatically set unknown transcripts or to manually set selected transcript For the first option unknown transcripts are set automatically Note No transcript can be set in case the mutation was found in orders with different transcripts in case the gene file was deleted or mutations were not found in real orders e g imported mutations For the second option you can select a transcript that should be set from the genome or gene file In case new mutations were added already before setting transcripts for the old entries mutations can be merged after transcript IDs are set To merge all mutations for one or several selected open the context menu in the Gene table and select merge The following options are available in the field Operation merge unknown transcript in case equal mutations are present one with a transcript and one with an unknown transcript the mutations ar
48. e merged Only one entry remains inclunding all information such as Orders Frequency of both previous entries merge equal transcripts mutations are only merged in case they are equal also the transcript has to be equal By pressing OK transcripts are set and depending on your settings mutations are merged After merging you will get a message showing how many mutations were merged Moreover you have the option to merge two mutations in the Mutation table Therefore select the mutation s in the Mutation table open the context menu and select merge mutations gt You can select which mutation should be added to the other one Index context menu This will only make a difference in case columns like AA Name are not equal In case the column Transcript is unknown or empty for one mutation and the other mutation has a known transcript the transcript will always be set during merging 2 3 2 SeqType genomic or cDNA For each mutation it is listed in which SeqType genomic or cDNA the mutation was detected Therefore the new column genomic cDNA of total is present in the Mutation table For all newly added mutations the count in this column is increased automatically Moreover the SeqType information is available in in dialogue Mutation in the field g cDNA For mutations that are present in the database already the following options are available to set the What s new SEQUENCE Pilot 4 2 0 8 count in column genomic cDNA o
49. e number of positions with a coverage warning expected quality no call required o AvgCoverage average coverage fwd rev o AvgQuality score average quality score fwd rev o The section Adaptors is not available anymore Adaptor trimming is done before reads are mapped to ROls Therefore this information is now available in the Info Window operation Sequence Files context menu show Info o n section Amplicons all amplicons are listed with the following information location direction average coverage fwd rev average quality score fwd rev coverage warning number of aligned reads number of positions with a coverage warning expected quality no call required o n section Unused Reads the number of unused reads is listed These are all reads that were mapped to the ROI but not aligned The error code number is written in front You can open a list of all possible error codes in the Menu Help item UnusedReadsErrorCodes e New context menu show gt unused reads A new window opens that lists the reads that were not aligned The same information is also present in the UnusedReads txt file Note Reads that were not mapped to the ROls are not listed here They can be displayed using the context menu item show unused reads in section Files Note Reads that were mapped to an homology region of the ROI are not shown by default But if you do a search for Error code Error4 the corresponding reads are diplayed
50. ected ROIs is available to e remove ROIs copy ROIs Select Profile settings e setthe Analysis mode only mapping What s new SEQUENCE Pilot 4 2 0 44 e active inactive ROIs 9 2 Operation ROIs Group master file ROIs can be grouped This has the following advantages Groups can be selected more quickly in the operation Run Groups can be exported imported for exchange with other institutes or JSI For a Group a list of primer pairs can be added button Import Primer The primers are joined to the corresponding ROI and amplicons are generated automatically entered in the section Amplicons PCR Primers in ROIs master file To create a Group Enter a group name in the field Name Move all ROIs to group to the ROI Group List using Add gt Press Save 9 3 Operation Run 9 3 1 Data lon Torrent as well as paired end sequencing data e g MiSeq can be analysed now 9 3 2 Multiple Processing Cores To make analysis faster multiple processing cores can be used to compute the result files of a run Several worker processes can be started which process several files in parallel In case the Run is started for several files each worker processes one file The number of worker processes should be related to the number of cores available on the server To define several worker processes Create the file RemoteComputer txt in the bin folder of your SEQUENCE Pilot installation Write into the
51. en many patients are loaded in one Autorun file and no validated controls are present All samples are regarded as control each sample is compared to all other samples New buttons Import and Export Analysis modes CNV can be imported and exported respectively sae file In case several analysis modes are defined for one ROI Group they can be grouped For grouped analysis modes the results CNV table and diagram can be shown together in the CNV window operation Sequence To group several analysis modes the button Grouping is available If this is pressed a new window is opened to group analysis modes In case an ROI Group with a defined Analysis Mode CNV is exported in operation RO Groups master file button Export the grouping information is exported as well 6 14 3 Operation Joining With the context menu item edit Patient Type in the Lower table the type patient control can be changed for one or several files In case a control that is already joined to patients CNV window is set as patient the control gets a pink background in the Controls list CNV window 6 14 4 Operation Sequence CNV window Controls section already joined controls that were set to type patient afterwards are marked pink To remove them press Control setting and then OK Button Control Settings New tab Run Here all control that have the same AutorunlRunlD are listed The same RundlD is present for o all samples that we
52. eral times In case the mapping to the genome is missing or another error occurs an according message is shown reads FASTQ reads FASTQ Creates one FASTO file for the comple file The nfo window context menu show gt info was adapted In section Settings the name of the Profile is listed In section Filter the name of the filter and filter settings are listed In section Trimming information the number of processed reads trimmed reads and discarded reads is listed 6 12 5 ROIs Location The Selected ROI can be exported as BAM or FASTQ files Therefore use the context menu item export What s new SEQUENCE Pilot 4 2 0 31 For both exports the following is done A directory to save the data can be selected Alternatively you can create a default directory that always should be used To use this option enter the path of the export folder in the 1is ini file section SegPilot behind the entry ExportDir When the export is finished the number of generated files and the path of the export folder is shown The file name includes DNA number o reads BAM creates a BAM file and a BAM BAT file for the selected ROI The same information is present as for the BAM export in section Files o export gt reads FASTQ creates a FASTO file for the selected ROI The context menu entry show gt info changed o In the section General the following additional information is shown o CoverageStats shows th
53. es are not loaded 4 Module SeqPatient 4 1 Import of Variation DB files ShPinown mato orar ave again now drabase Fave o be rad as dose A database package is available to install the following databases dbSNP Short Genetic Variations http www ncbi nlm nih gov SNP 1000 Genomes Catalog of Human Genetic Variation http www 1000genomes org ClinVar Sequence Variation and its relationship to human health http www ncbi nlm nih gov clinvar CLINVITAE Clinically observed genetic variants http clinvitae invitae com COSMIC Catalogue of somatic mutations in cancer http cancer sanger ac uk cancergenome projects cosmic These can be used to show known SNP information and for filter options see operation Sequence For the genomes available on our homepage we offer an exe file for easy installation of all Variation databases There are two installation packages we offer for download one referring to hg19 the other one referring to hg38 please select the correct one for your installation depends on the installed genome hg19 or hg38 there is also the possibility to install both packages at the same time please keep in mind that in this case both genomes hg19 and hg38 have to be installed The database package can either be downloaded from our homepage or alternatively from our ftp server We recommend to use the ftp server for the download of big files because the download can be restarted in case t
54. ew SEQUENCE Pilot 4 2 0 22 6 8 Operation ROI Groups master file Button Export The following new option is available when an ROI Group is exported o The Group name lot number can be replaced Therefore enter the new names into the fields Group Name and Group Lot respectively o The panel name can be replaced Therefore enter the new panel in the field Panel o The suffix name can be replaced Therefore enter the new suffix in the field Suffix Button Import the following new options are available when an ROI Group is imported o The Group name lot number can be replaced Therefore enter the new names into the fields Group Name and Group Lot respectively o The panel name can be replaced Therefore enter the new panel in the field Panel o The suffix name can be replaced Therefore enter the new suffix in the field Suffix o n case gene files from Gene Admin should be used as references activate the box map to gene files Gene Admin In this case the dialogue Change Gene Transcript opens Here you can adapt the transcript that should be used as reference After pressing OK you get a message showing how many ROIs were mapped to a gene file Gene Admin For ROIs that can not be mapped to a gene the gemome is used as reference Note The mapping to a gene file only works in case the ROIs were set up with a genome reference In case a the ROIs were set up with a gene file reference already no mapping to another gene
55. ew item edit gt is available for one or several selected resultfiles Here DNA number type gender and digested can be changed There is a separated sub menu present for each item o Settings Upper table is available for several selected files now Using this a new window opens to change the Mix for all selected files simultaneously 10 2 Operation Sequence Files table the new column MValidation is available Here the user and date of the medical validation is listed Report The sorting of the analysis mode methylation is now adjustable as in the analyse What s new SEQUENCE Pilot 4 2 0 53 diagram before it was sorted by fragment length 11 Talkmaster Modules SeqPatient SeqNext For calculation of the position of a mutation variation previous mutations e g insertions deletions are not regarded any more Each mutation variation is regarded standalone in relation to reference sequence e The fields Mark and MutDB in the Variation Mutation table can be exported Transcript information can be exported for each mutation The HGVS c and p nomenclature can be exported HGVSNucName and HGVSAAName As export format vcf is available now The following new items can be exported for module SeqNext Average coverages fwd rev for genes ROIs and amplicons e Number of variations for each result file for genes ROIs and amplicons Disease number MutID Module SeqPatient All information e g Fi
56. f checked no alignment evaluation is done The filters below are not used e Max 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 Compl reads only If checked reads that do not cover the complete amplicon are discarded Barcode 5 3 Choose this setting in case barcodes have to be present at both ends of the reads Ignore paired end info If checked paired end information is not used Skip reads If active only reads with a copy number that is higher than the entered Skip count original value take part in the analysis Here the original reads from the sequencer not the reads mapped to the ROI are used Note This will reduce your coverage What s new SEQUENCE Pilot 4 2 0 48 9 3 5 Start a run To start a Run manually Press in the section File to select your file s e Optionally change the Settings Add DNA nu
57. f total for already existing orders Note It is recommended to set the SeqType for all entries in the mutation database You can set the SeqType for all mutations of one or several gene s Therefore select one or several gene s in the Gene table open the context menu and select set genomic cDNA You can set the SeqType for one or several mutations Therefore select the mutation s in the Mutation table open the context menu and select set genomic cDNA For both options the following operations are available 2 3 3 automatically set genomic CDNA the program checks which orders are present for genomic and cDNA sequences and sets the count automatically manually set all to genomic all orders are expected to be genomic data the genomic count is increased depending on the number of orders manually set all to cDNA all orders are expected to be cDNA data the cDNA count is increased depending on the number of orders Select Mutations When one or several genes are selected in the Gene table by default all mutations for these are shown in the Mutation table This new section is available to search for mutations using special search items If Search is pressed only mutations that fullfill the search items are listed in the Mutation table The following search items are available Transcript Only mutations of the selected transcript are listed Location Only mutations of the entered location are listed Mut Effect Onl
58. file is used What s new SEQUENCE Pilot 4 2 0 27 6 10 Operation Joining Poollist In the Lower table Runs table the patient type patient control can be edited therefore select one or several files and use the context menu edit Patient Type A list opens where the type patient control can be changed In case a control that is already joined to patients CNV window operation Sequence is set as patient the control gets a pink background in the Controls list CNV window 6 11 Operation Joining New column Warning in the Upper Table Here a warning can appear in case the transcripts that were used to create the ROIs can not be found in the downloaded genome This can only be possible in case ROls ROI Groups from another SEQUENCE Pilot installation were imported Import button in RO ROI Groups master file the genome was replaced by a new genome containing other transcripts e g hg19 with ENSEMBL references was replaced by hg19 with Genbank references In this case please correct your ROIs in operation RO master file The following context menus changed in the Upper table show info In the Info window the following new information is shown o n section Settings the name of the Profile is listed o n section Filter the name of the filter and filter settings are listed o n section Trimming information the number of processed reads trimmed reads and discarded reads is listed e Settings
59. file on which IP port to reach the remote computers e g 127 0 0 1 7301 127 0 0 1 7302 127 0 0 1 7303 127 0 0 1 7304 Save the file and start SEQUENCE Pilot as usual Now several worker processes are available automatically in the example above 4 worker processes are started They can be seen in the task manager 4 entries for SeqNResultfilesWorker exe They all quit automatically as soon as one quits SEQUENCE Pilot 9 3 3 Importer By default the Importer uses the maximum number of cores Therefore the computer might be very slow during data is imported which might be a problem in case other programs are used as well The number of cores used during import can be restricted in the 1is ini file located in the bin directory of your SeqPilot installation Therefore make the following entry in the section SeqNextHLA MaxImporterThreadCores 1 What s new SEQUENCE Pilot 4 2 0 45 The number behind is the number of cores used for import 9 3 4 Settings Adjustable settings for each analysis are available Settings can be saved as Profiles If the as default option is activated for a Profile the profile is used automatically when a Run is started unless another Profile Settings are selected manually The following settings can be adapted Tab Settings o Reads 1 HT Basecalling Basecalling coverage Decides if a second base is called or regarded as background The second bases has to reach this percentage
60. for Homologies name of the ROI chromosome range Moreover a list of unused reads can easily be opened in operation Sequence Therefore use the context menu item show gt unused reads in section Files 6 6 3 Tab add PCR and tab add Panel In the field Suffix special characters can be entered to automatically define the ROI name The default ROI name is GENE LOC suffix o GENE Gene name o LOC Location Exon name o additional characters are TID or ISO transcript ID Example for BRCA1 ENST00000001 E10 e In the Suffix field the following entry is listed Test GENE TID location LOC end o The following ROI name is created Test BRCA1 ENST00000001 location E10 end 6 6 4 Tab add PCR tab Gene The following entries are available in the field Organism gene file genome the gene will be searched for in all loaded genomes unless the gene file was loaded Gene Admin In this case only the loaded gene will be shown The column Source shows the source of the gene genome or gene file gene file the gene from gene files Gene Admin will be used What s new SEQUENCE Pilot 4 2 0 20 genome e g hg19 the gene will be searched for in the selected genome The new field Chromosome is available When a chromosome is selected here only genes located on this chromosome are available to create ROIs 6 6 5 Tab add PCR tab Fasta In case fasta files are used for ROI set up they have to locate in the dire
61. for Seg Type genomic intron sequences in between are included for SeqType cDNA only exon sequences are analysed When an exon is selected in the table the location is entered into the field below automatically When several exons are selected in the table both exons are used including the intron in between New field SeqType here you can select if you analyse genomic or cDNA For cDNA no introns are included What s new SEQUENCE Pilot 4 2 0 41 Grouping of SeqPrimers is possible For grouped primers results can be shown as one sequence in operation Sequence select the PrimerGroup in the section Group To group SeqPrimers first add a PrimerGroup in the section SegPrimer group Then select the group in the field Primer Group of the SeqPrimer table for several SeqPrimers 8 4 Operation Joining A faster algorithm is used therefore the processing is faster 8 5 Operation Sequence A location overview is shown above the electropherogram introns are marked yellow exons are marked blue Below the overview the length of the each resultfile is indicated Forward sequences are shown in a lighter color as reverse sequences There is a context menu available in the Location overview o show peak height ratio Below the location overview a peak height ratio diagram for each base position is shown The bars show positions with a high background In case a background is present there is a bar above the line for forward b
62. g the context menu show fragment size in the coverage graph Location overview o same dir pair Only present for sheared paired end data without amplicons e g Haloplex data The setting randomly sheared has to be set in the Settings operation Run This warning is shown in case one read of the read pair has changed the direction This might be a hint for a possible inversion When you select the mutation in the Variation table it is jumped to the position where the read pair starts in the sequences In column Position the sequence range showing mismatching positions is listed o swapped pair orientation Only present for sheared paired end data without amplicons e g Haloplex data The setting randomly sheared has to be set in the Settingsloperation Run This warning is shown in case the reverse read lies in front of the forward reads This is a hint for possible translocations 6 12 7 3 Variation DB display and filter function Imported SNP DBs can be used for display and filtering Therefore the context menus show table DBs and filter gt DB are available in the Variation Mutation table Show table DBs Information for mutations present in the gene files and mutation databases can be shown in the Variation Mutation table and sequences One tab is present for the gene file in case a gene file from Gene Admin is used as reference sequence and for each imported SNP database The number of Variations Mutations described in the database
63. ges or a base change and an insertion deletion to consider them as an ndel is 3 This gap can be changed in the 1is ini file located in the bin directory of your installation Therefore enter the number of bases behind the following entries in section SeqPilot o for base changes Del InsGapSNPToSNP o for base change insertion deletion Del InsGapSNPToInDel Note This setting can also be changed for each gene separated Therefore use the context menu editing gt InDel gaps in the section Genes Imported SNP DBs ClinVitae COSMIC ClinVar 1000 Genomes dbSNP can be used for display and filtering The following context menus are available for filtering and display o show table gt DBs Information for mutations present in the databases can be shown in additional columns of the Variation Mutation table o filter gt DB Mutations can be filtered For filtering special internal filters can be used as as well as imported databases The new tab filter is available it lists mutations that were filtered using the SNP database filter Mutations on this tab are not listed on tab all It is possible to print this tab on the Report by selecting the tab filter in the Print Preview Please have a look at our User Manual SeqPatient for detailed instructions What s new SEQUENCE Pilot 4 2 0 14 The following new context menu items are available o ignore toggle If selected the mutation will be ignored and not
64. heck edited het pos and mism e all it is jumped to exon and intron positions In case introns are sequenced they can be saved in the HLA DB for result alleles with unknown intron sequences Therefore use the new context menu item intron save as in the Haplotype sequence Matching tableltabTotal Result context menu show sequence specific primers primer with lot numbers can be required What s new SEQUENCE Pilot 4 2 0 42 9 Module SeqNext HLA The module has been completely worked over the functions are similar to the corresponding functions in the module SeqNext With the new version lon Torrent as well as paired end sequencing data e g MiSeq can be analysed 9 1 Operation ROIs master file Note HLA Kits from the previous versions are not valid anymore ROIs have to be newly defined using the procedure described below Intronic regions can be defined Note For intronic regions the sequences are only visualized but not used for result calculation For result calculation only exons are used Introns can only be defined together with exons Moreover only reads that cover a part of the exons are aligned Reads that cover intron areas only are not aligned Therefore for big introns there might be a drop in coverage in the middle of the intron The only positions that are evaluated in the intron are the splice sites first two bases and last two bases of the intron If a mutation is found here the warning X is
65. here is an interruption Download from our homepage Go to http www jsi medisys de genomes snp dbs Download the file hg19 GenomeVarDB and or hg38 Genome VarDB After download please verify the integrity of the downloaded file i e whether it is complete and uncorrupted To do so you may use the md5 checksum provided to the right of the respective link Search for md5 checksum on the web to find suitable verification tools Within the verification tool browse and select the downloaded file and let the tool compute the checksum Compare it to the one provided on our homepage If the two checksums differ you have to download the file again Download from our ftp server You can reach the file server using the following link ftp ftpsrv jsi medisys de What s new SEQUENCE Pilot 4 2 0 11 e Please use the following login Username SeqPilotData Password SeqPilot Download the file hg19 GenomeVarDB exe from folder GenomeVarDB hg19 and or file hg38 Genome VarDB exe from folder GenomeVarDB hg38 The md5 checksums provided with the download links on the website may be used for the respective file on the file server too see above To install the databases Execute the file hg19 GenomeVarDB exe and or hg38 GenomeVarDB exe and follow the installation instructions As destination directory C SeqPilot GeneFiles is proposed automatically In case this is not the folder of your SeqPilot installation please
66. in the tool tip of HT1 HT2 and combined sequence Coverage This is the number of called bases at a position e 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 Moreover quality scores are shown as a color code behind each base The following colours are used by default o quality score 1 10 Dark Red o quality score 11 20 Red o quality score 21 30 Yellow o quality score 31 40 Light Green o quality score 41 99 Dark Green HT1 and HT2 Another allele can be entered into the haplotype field When the button is pressed the green HT sequence changes to the entered allele sequence and the alignment to this allele is shown There is the new count mode ROI abs available In this count mode the first position of the ROI is 1 What s new SEQUENCE Pilot 4 2 0 51 9 5 4 Reads sequences The marking all reads with a perfect match to an allele is not available any longer by default To get the mark select Mark perfect matches count in the Settings when you start the Run Context menu entry set fragment as HT1 set fragment as HT2 For paired end data you can decide if you want to set the selected read only as haplotype or the complete pair Therefore you can select read or pair Note This option is only available for pairs 9 5 5 Reads view New field Quality If this field is activated quality scores are shown as a color code bel
67. jectSelect can not only be set in section LIS but for all modules separated now With this entry a project can be pre selected in the section Select Orders of Joining Worklist and Orderlist Therefore make the entry into the section of the module in the 1is ini file This is SeqPilot for modules SeqPatient and SeqHLA SeqNext for module SeqNext SeqNextHLA for module SeqNext HLA 1 4 Menu System Protocol Manager Using the Protocol Manager you can select what is shown in the Protocol All events that can be shown are listed in the column Events There are several columns that can be changed for each event Protocol on off event is listed not listed in the Protocol respectively Show default on off All events that are default events can be searched for in the Protocol When the Protocol is opened in the section Select Protocol the event default can be searched for When this is set only events that are set to Show default on are listed in the Protocol Show protocol tab on off event is listed on the tab Protocol operation Sequence Colour R G B the event can be highlighted in color By default the events set delete order state MV set delete gene state MV and change base are highlighted Tooltip A tooltip for the event can be shown in the tab Protocol operation Sequence By default a tooltip is shown for the event change base e Shortcut In this column a shortcut for an even
68. ld of the section Line o Enter the line number in the second field of the section Line o 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 are listed in a new window The line information is available in the tooltip of the read when org read is active New button Filter Here you can filter for reads that fulfil special criteria When OK is pressed only those reads will be displayed in the reads view Context menu entry set fragment as HT1 set fragment as HT2 For paired end data you can decide if you want to set the selected read only as haplotype or the complete pair Therefore you can select read or pair Note This option is only available for pairs 9 5 6 Matching table There is the new column S Factor Sorting Factor available This factor sorts the alleles by their probability The smaller the number is the higher the allele combination is listed It is calculated the What s new SEQUENCE Pilot 4 2 0 52 following way number of mismatches divided by number of known exons for the allele Example Two possible allele combinations show 1 mismatches For allele combination 1 allele sequences are known for 4 exons S Factor is 0 25 For allele combination 2 allele sequences are known for 2 exons S Factor is 0 5 The allele combination 1 is listed first in the Matching table 9 5 7 Show New jumpers in the check box
69. lename Primer SeqGeneparts can be exported per variation now n case several result files per mutation are listed comments TV and MV User date etc can be exported as well The following new items can be exported for module SeqNext HLA Average coverage per ROI e Projects Comments e Warnings Module MLPA The copy number changes are exported in the same sorting as in the analyse diagram before they were sorted by fragment length What s new SEQUENCE Pilot 4 2 0 54
70. master file for the amplicon These columns are not shown by default To show them right click the table header and select Manage table columns Increase the column Width e g 20 6 12 7 Variation Mutation table 6 12 7 1 Mutation Calling of Insertions Deletions and Indels overlapping an ROI Rules for calling of insertions indels and deletions that reach over the beginning end of an ROl amplicon An insertion is called in case both flanking bases lie within the ROI amplicon What s new SEQUENCE Pilot 4 2 0 33 e exception in case only one flanking base lies withing the ROl amplicon mutations are only called when o the ROI is extended o primers are defined for amplicons Note the mutation must start after the primer and reach into the amplicon A deletion indel is called incase it completely lies in the ROl amplicon e exception the mutation begins ends in the ROl amplicon but reaches over the border Mutations are only called when o the ROI is extended o primers are defined for amplicons Note The mutation must start after the primer and reach into the amplicon Note For restricted ROIs column Restrict in ROI master file was activated manually and amplicons without primers the following is called Deletions indels that reach over the ROl amplicon beginning end are not called completely Only the part that completely lies in the ROl amplicon is called Example A 5bp deletion is present only 2 bp lie
71. mber and optionally a barcode and project in the dialogue Patients Select ROIs or an ROI group for one or several selected patients Press Analyse Automatic Start of a Run You also have the option to start a Run automatically With this option all parameters needed for the Run have to be written in a txt file This txt file is used to start the run instead of entering all parameters in the operation Run manually The file can be created using any text editor and has the following format All fields have to be separated by tab several entries in one field by semicolon In case one field is not filled out it has to be separated by tabs anyway 1 DNANo Barcode optional ROI or ROI Group Path of the file s Profile Project NOOR WN Resolution 4 digits 2 digits max In case several files patients are analysed copy the first entry into the next line and adapt it Each line stands for on Run to be started Copy the txt file into the folder bin Autorun SeqNextHLA of your SeqPilot installation The runs are started automatically and the txt file is moved to the backup folder 9 4 Operation Joining The new search field HLA Group is available to search for orders using a certain HLA Group With the context menu entry settings in the Upper table the Settings Profile adjusted in the operation Run can be changed In case the settings are changed the file is recalculated automatically using the new settings After
72. moved in the pool view with the option pool patients the mutation is moved in the pool view and for each order e Technical and medical validation buttons TV and MV the dialogue where the user can decide if TV MV is set for the pool only or for pool and all Patient orders changed You have the options pool only pool patients and patients only to define what should be validated 7 Modules SeqHLA and SeqNext HLA 7 1 Importer Identical file names can be used now The files are identified by the file date 7 2 Menu SeqHLA SeqNext HLA This database includes exon as well as intron sequences Intron sequences can only be visualized in software version 4 2 0 for previous versions they have no function HLA DB Admin o An HLA database including intron sequences is now available for installation Note For intronic regions the sequences are only visualized but not used for result calculation For result calculation only exons are used Introns can only be defined together with exons The database is available on our homepage http www jsi medisys com hla database Download the exe file that includes intron sequences To install the database close your SeqPilot installation Start the exe file and follow the installation instruction Make sure to enter the directory of your SegPilot installation during installation by default C SeqPilot is used In the dialogue HLA DB Admin the new field Show is available Here you ca
73. n combination with paired end reads This setting is optional If active the fragment size is analysed which gives a hint for big insertions deletions Force combined coverage This setting is useful when What s new SEQUENCE Pilot 4 2 0 25 o per dir settings and or ratio read direction settings are used in sections 2 Required coverage or 4 Mutations of tab Settings In case these settings are not used the force combined coverage is greyed out o mutations with a high coverage are present in any sequencing direction but are not called because settings in sections 2 or 4 are not fullfilled o n case the coverage of the mutation reaches the Force combined value in at least one sequencing direction the following settings are changed automatically In section 2 Required Coverage and in section 4 Mutations all per dir settings are switched to combined and all ratio read direction settings are switched to off o The mutation is called in case all settings are fullfilled now mutation calling is done using the new settings It is marked pink in the Variation Mutation table to indicate that the combined mode was used automatically In addition the following options can be activated o Only 4 For the new calculations the combined mode settings are only used in section 4 Mutations The settings in section 2 Required coverage are not changed o no WT the Force combined setting i
74. n select if Exon or Intron sequences should be displayed when button show alleles is pressed Intron sequences are only available in case a database including introns is installed e HLA DB Update This menu does not exist anymore n1u files for database updates are not available anymore Please use the exe file provided on our homepage http www jsi medisys com hla database for database updates New item HLA allele comparison compares alleles of a certain gene The sequences of both alleles are shown as alignment Heterozygous positions are shown below What s new SEQUENCE Pilot 4 2 0 40 7 3 Operation Archiving When orders are archived entries with mismatches gt 0 are deleted in the tables RFmatch and RFmatchDetails 8 Module SeqHLA 8 1 Operations Amp modules master file SeqPrimer master file Sequence Intronic regions can be shown Note For intronic regions the sequences are only visualized but not used for result calculation For result calculation only exons are used Introns can only be defined together with exons The only positions that are evaluated in the intron are the splice sites first two bases and last two bases of the intron If a mutation is found here the warning X is shown in the column Warning of the Positions Resultfiles section The splice site mutation is not used for result calculation 8 2 Operation Amp modules master file The following fields changed
75. n the file server too see above To install the databases Execute the file hg19 GenomeVarDB exe and or hg38 GenomeVarDB exe and follow the installation instructions As destination directory C SeqPilot GeneFiles is proposed automatically In case this is not the folder of your SeqPilot installation please change the link to the GeneFiles folder of your SeqPilot installation What s new SEQUENCE Pilot 4 2 0 16 Note In case you do not want to create the database folder in your SeqPilot installation but in another directory on your PC you have to enter the path of the directory in the 1is ini file section SeqPilot GenomeVarDir Path e g C GenomeVar GenomeDBVarDir Path e g C GenomeDBVar 6 3 File UnusedReads txt All reads that were neither mapped nor aligned are written to a file The files UnusedReads txt are generated for the complete File and for each ROI Complete File The UnusedReads txt locates within the folder SeqNResults in the corresponding year month and Run ID folder The same information can be opened in operation Sequence Therefore use the new context menu item show gt unused reads in section Files ROI For each ROI all reads that were not aligned are written to the Unsused Reads txt file It locates within the folder SegNResults in the corresponding year month Run ID and ROI folder The same information can be opened in operation Sequence Therefore use the new context menu i
76. nce Variation Mutation table The new column Transcript is available which lists the used transcript For the VCF export context menu entry export gt tab to VCF you can select if you want to export the file for CARTAGENIA or for other platforms Therefore a new window opens where you can select a path for the output file field Output and the types General or CARTAGENIA field Type For big insertions deletions gt 30 bp all mutation data is available The mutations can be added to the mutation database HGVS nomenclature o The column HGVS nomenclature was renamed into c HGVS o The HGVS p nomenclature is shown in the new column p HGVS In case the NucName and the AAName given in the Mutation database differs from the entries in column c HGVS and p HGVS the entries from the mutation database are listed in the field mut Entry in parenthesis For mutations with the Hint not detected no zygosity is shown in column Nuc change Report For calculation of the position of a mutation variation previous mutations e g insertions deletions are not regarded any more Each mutation variation is regarded standalone in relation to reference sequence 2 3 Operation Mutation master file 2 3 1 Transcript ID The transcript ID is now available for entries of the Mutation database For all newly added mutations the transcript ID is listed in the column Transcript automatically Moreover the field Transcript is availabl
77. ng entry is present e g chromosomal start end position is wrong or identical chromosomal positions are present in several lines Hint In case the chromsomal position in the line was translated into an ROI the Gene Exon Location genome and transcript is listed 6 7 Set up of Multiplicom MASTR assays Multiplicom MASTR assays are not set up using tsv files any more This does not affect ROIs created with tsv files in previous versions Multiplicom offers special bed files for SeqNext that can be used for import These can be imported in ROI master file tab add Panel Note Please make sure to download the special bed files for the software SeqNext from the Multiplicom homepage Otherwise the primer information will be missing In operation ROI master file tab add Panel the following is recommended for Multiplicom bed files Dialouge Import Panel File For MASTR assays without copy number variation analysis please change the following fields o Sense Primer 7 o Apntisense Primer 8 For MASTR assays including controls for copy number variation analysis please change the following fields o Comment 4 o Sense Primer 7 o Antisense Primer 8 o CNV Control 4 o Key Control Section Settings Make sure to check the box build amplicons In case gene files from Gene Admin should be used for the setup activate the option map to gene files Gene Admin Note The genefile has to be mapped to a genome What s n
78. ng the mutation is shown in a new line below the gene line after OK is pressed Indel Here the base sequence of an Indel or the number of inserted deleted bases can be entered The resulting sequence including the mutation is shown in a new line below the gene line after OK is pressed o The context menu item Heterozygote start toggle is not available anymore New context menu item show family for the result file sequences shows all result files belonging to one family in the new result file view The following new count modes are available in the combo box count mode o Gene abs absolute position in the gene o Genome Position genomic position 4 4 2 Genes The following new context menu items are available editing gt o frame shift analysis on off Use this to switch the frameshift analysis on or off If switched off in case of a frameshift each position is regarded separated many heteroygous What s new SEQUENCE Pilot 4 2 0 13 positions will appear Note In case the frameshift analysis algorithm was used automatically there is the condition F in the column Condition section Position Resultfiles o Indel Gaps The new mutation type ndel is present in the Variation Mutation table By default the maximum number of bases between two base changes or a base change and an insertion deletion to consider them as an ndel is 3 This number can be changed using this context menu 4 4 3 Positions Resultfiles
79. nly saved in case they enclose an existing amplicon Otherwise the primers are not imported you will get a list of invalid primers and possible ROIs No new additional amplicons are added When an ROI Group is deleted you can optionally delete the ROIs belonging to the Group The section CNV Group to select control samples for CNV analysis is not available in this operation any more It was moved to operation Analysis Mode master file What s new SEQUENCE Pilot 4 2 0 23 6 8 1 Import of Pseudogenes from Pseudogenes org Pseudogene lists from Pseudogenes org http pseudogene org can be imported to filter pseudogene reads A list of human pseudogenes for import can be downloaded Please note the latest pseudogene files correspond to genome hg38 NCBI38 In case you use genome hg19 NCBI37 please use Pseudogene release 74 or older To find all Human Pseudogene Releases do the following Go to http pseudogene org Human Press the link behind Other Human Pseudogene Sets A list of Human Pseudogenes opens Scroll down to find Human Pseudogenes Build 74 Press download behind this entry Note For server installations the Pseudogene org file must be placed in the directory defined as NewResultFiles directory for module SeqNext This folder is specified in the 1is ini file bin directory of your SeqPilot installation behind the entry NewResultFiles in section SeqNext For single user installation
80. nton educere Eben Ox ER Pe E c OR REO nu KR ERE DER EA DERE ELE PEE DELIA DAT TTA R 7 2 3 Operation Mutation master file ssssessesssssessssssseseeeeeneennnenn nnne nnne nnean 7 231 Transept ID eem MINE RERO Pe PER Fede Nep 7 204 CHO ype genomic Or CUNA i pedi decptesitet s nt d Pd edi EE DAtUME LM d NN pb MEME EePPP S p a aM MEE UpEN 8 pese cce II dcr c DE AN 9 px nr di Ri et A ac E E E N E E EE yee 10 9 Modules SegPatient and Seghlbi usciivexixsav Kn tappe EDRAYURIE HE SEn pRa CELER REDE EK AISEE 11 S1 D DERBI JOU ossi duc aen iiec aA ipa nial aT ERU E ADS UR d kPa ON pni I ON 11 d Module dev ect ET EE DEED ES 11 4 1 Import of Variation DB files ssssssessssssssssssssssssseeeeeen enne nenne nn nnnm enirn nni 11 L2 Gene Jb nici pibe eR e TE LR OU E NEU ORI HE US GUN ERROR E QUA DURO NEU EMICRURDE ORA RR 12 xS Nt M 12 m4 tperSan Sedul S ooi oe nau IO tr ee ea eai ete unu s nds iun cere en re ee s eria eds 12 Lo Pul secs de PITT Tm 12 LA UN EC PETENTEM MT 13 4 4 3 Positions Fesultfiles ilinilcun eec seu a epa tran Arena Re Rak o au kr rac Rr RR rin R A pan E ae RR nS Ad EEEa 14 4 4 4 Variation Mutation table ccccceeceeeeeeeeeeeeeeeeeenaeaaaaaeaaeeeeeeceeeeceeeeeeeeesaeaeeeseaeneeeesaanees 14 4 5 Operation Mutation master fe esiseina PEDE EO UU PU EH E RES misao iaa Mdb p 15 5 Modules SegMext and SaqNext ALA isis uii seerraxsieerk
81. number of reads with a perfect match is below this value complete seq Warning F in case the number of reads with a perfect match and that cover the ROI completely is below this value Note This option does not work in case amplicons are defined Allele 1 Allele 2 proportion Warning A in case the ratio of allele 1 to allele 2 coverage is below this value Two options can be additionally set for this setting perfect matches only only the reads with a perfect match to the called alleles are regarded for calculation of the ratio complete seq only reads with a perfect match to the called alleles and that What s new SEQUENCE Pilot 4 2 0 46 cover the ROI completely are regarded for calculation of the ratio Note This option does not work in case amplicons are defined Basecalling coverage background Warning B in case a third allele contamination or background exceeds this value for at least one base position You can choose if this warning should be calculated combined or per Haplotype Use the jumper bg BC in the dialogue Show to jump to the corresponding positions Basecalling Indel background Warning N in case the coverage of an insertion deletion exceeds this value You can choose if this warning should be calculated combined or per Haplotype Use the jumper bg Indel BC in the dialogue Show to jump to the corresponding positions DRB pseudogene plausibility check Warning P in case DRB1 expected pseudogenes
82. o MR uem 25 CAPUAE MORE UU UU UTEM 27 Eee il yj Hp st MEN NR NEN 27 What s new SEQUENCE Pilot 4 2 0 2 6 10 peer one Dp iure NR E ne ecccuecteecteiatendentaddapaararneeetasele 6 11 Operation Joining T 6 12 Operation Sequence 6 12 1 SUERTE po dd j 6 12 4 ee 6 12 5 ROls Location ROCCO RUPTURE TEC TEMPI COT PROPRE NER OT RN PRO ES ANS 6 12 6 Summary 6 12 7 Variation Mutation table 6 12 7 1 Mutation Calling of 6 12 7 2 Tabs Me 6 12 7 3 Variation DB display a and filter function Mes dx did DUE D 6 12 7 4 Context menu 36 6 12 8 ee EC Oe ore eee 6 12 9 Show 6 12 10 Report 6 13 Operation Arc 6 14 CNV analysis een N ET S P PU re 6 14 1 Operation ROI master file d T Mn 6 14 2 Operation Analysis mode CNV m ster r file 6 14 3 Operation Joining EEE E E E PETNE 6 14 4 Operation Sequence CNV window huecos LE Shas tala EL E AEL 6 15 Operation Pool i ETA 7 Modules SeqHLA and SeqNex HLA 7 1 Importer 7 2 Menu SeqHLA SeqNext HLA 7 3 Operation Archiving m 8 Module SegHLA N EEOSE i c EM en eee 8 1 Operations Amp modul i master file SeqPrimer master file Sequence 8 2 Operation Amp modules master file 8 3 Operation SeqPrimer Lape file 8 4 Operation Joining 8 5 Operation Sequence 9 M
83. odule SeqNext HLA 9 1 Operation ROIs master file sin 9 2 Operation ROIs _ neil inen E E AA NM RT 9 3 Operation Run ee ee eer re eee 9 3 1 Data 9 3 2 Multiple Proc Core 9 3 3 Importer DERE E o MET was Gen at errors 9 5 ipii Eu ami A ee eee er een eee eee ECE tye See eee eRe ee 9 S 1 jes saan and Ei aes dae AE eae 9 a 3 Gambinad HT aud m sequen e 9 5 4 5 Reads sequences What s new SEQUENCE Pilot 4 2 0 3 1 All Modules 1 1 64 bit database All SEQUENCE Pilot modules and processes can be provided as 64 bit version now Note For server installations please contact our support team before updating your system to use the 64 bit version For single user installations To use the 64 bit version you have to change the link of the SEQUENCE Pilot desktop icon Therefore go to the bin directory of your SEQUENCE Pilot installation by default this is C SeqPilot bin Create a desktop link for the file SeqPilot64 exe When the new desktop icon is used the 64 bit version starts automatically Otherwise with the default desktop icon the 32 bit version is started In case you do not want to use the 32 bit version anymore please remove the default desktop icon 1 2 Automatic Timeout For server installations Standalone 0 there is an automatic timeout after 15 minutes in case no other timeout is defined in the lis ini file 1 3 Lis ini The lis ini entry Pro
84. ossible deletion fragment size possible insertion is shown The fragment size can be checked using the context menu show fragment size in the coverage graph Location overview orientation Only present for sheared paired end data without amplicons e g Haloplex data The setting randomly sheared has to be set in the Settings operation Run This warning shown that anomalous pair orientations are present This could hint at structural events like inversions translocations etc Hint in the Variation Mutation table is same orientation pair or swapped pair orientation Same dir pair One read of the read pair has changed the direction This might be a hint for a possible inversion swapped pair orientation The reverse read lies in front of the forward reads 6 12 3 Files Genes Chromosomes and ROls Locations The context menu item editing gt original is not available any more 6 12 4 Files New context menu items show e sequence Here a sequence can be searched for in the file Therefore o o Enter the sequence in the field Seq Check both directions in case the sequence should be checked in forward and reverse reads Optionally enter a gene name in the field Gene If this is done the sequence is only searched for in that gene Optionally enter a ROI name in the field ROI If this is done the sequnece is only searched for in that ROI Check only mapping to get the result for mapped reads only not for aligned
85. ow the bases The same color code as in the tooltip of the allele sequences is used New check box 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 that lists the read is shown as read identifier 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 a 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 in a new window The sequence that was searched for is highlighted To search for a read listed in the Next Generation Sequencing File o Select the file in the first fie
86. printed in the report The line will be highlighted grey You can remove the ignore by selecting the item again o move to other Is available for distinct mutations only Moves the mutation to tab other o move to distinct Is available for other mutations only Moves the mutation to tab distinct o export gt tab to VCF Note This only works in case the gene files Gene Admin were mapped to a genome prior to your analysis The selected tab is exported as a VCF file You can select if you want to export the file for CARTAGENIA or for other platforms Therefore a new window opens Output With the button you can select a path for the output file Type select the types General or CARTAGENIA Choose CARTAGENIA in case you want to import the vcr file into CARTAGENIA 4 5 Operation Mutation master file In case several isoforms are active and a sequence is present which does not belong to the main isoform the cDNA nomenclature is calculated based on the another active isoform 5 Modules SeqNext and SeqNext HLA 5 1 Automatic zipping of result data After starting a Run the data is zipped automatically This makes the result data much smaller approximately 10 of the unzipped result data 5 2 Operation Users master file The new user right User is authorised to edit profile settings is available This right is active by default In case it is inactive the user can not edit the Settings Profiles in the opera
87. re loaded in one Autorun File o all samples that were loaded in one Run The result table in the upper right part can be exported Therefore right click into the table and select export table from the context menu New section Analysis Modes Grouping In case several analysis modes are defined for one ROI Group they can be grouped For grouped analysis modes the results CNV table and diagram can be shown together in the CNV window operation Sequence To group several analysis modes in operation Sequence the button Grouping is available If this is pressed a new window is opened to group analysis modes The corresponding entries are then made in operation Analysis modes CNV master file automatically What s new SEQUENCE Pilot 4 2 0 39 The new table Analysis Modes Grouping is available in the CNV window To see the grouped view select the group name in this table To see the single view for each analysis mode select the entry Analysis mode CNV 6 15 Operation Pool e Variation Mutation table o New column Coverage Here the coverage forward reverse is listed In case the mutation was detected in several orders the average coverage is listed Context menu entry move to In case a mutation is moved from one tab to another e g move to tab distinct in the pool view you can decide if the mutation should be moved to the corresponding tab in the patient orders as well With the option pool only the mutation is only
88. rning is as listed below not analysed has the highest priority whereas expected has the lowest priority e not analysed An error occurred Please recalculate your file e dropout No sequences are available for more than 90 of the ROI No read are also present in case the Required Coverage Settings Min abs coverage and or Ratio read directions defined in operation Run section 2 are not fullfilled e pnocall There is no coverage at one or more positions of the ROI Only occurs in case no Required Coverage Settings are set o required This warning shows that positions in the file are ignored not analysed Positions are ignored in case the Settings for the Required Coverage Min abs coverage and or Ratio read directions defined in operation Run section 2 are not fulfilled The ignored positions are greyed out in the sequences forward reverse and combined sequence o quality This is shown in case the setting Low Quality score coverage warning operation Run is exceeded o expected this warning was named ow in the previous version The coverage of one or several positions is below the Expected coverage warning Min abs coverage default 100 The Min abs coverage is represented as a red dotted line in the electropherogram Moreover the Min abs coveage is shown in the graphical overview below the location overview The Expected Coverage warning Min abs coverage can be set in your Settings when starting the Run New
89. s e g useful for DRB If only DRB1 should be analysed you can define DRB1 DRB9 as ROIs Then select only mapping for DRB2 DRB9 in the ROI List The pseudogene sequences are filtered and not mapped to DRB7 In operation Sequence only results for DRB1 are shown Several ROIs can be set to only mapping in the ROI List by selecting the ROIs and using the context menu Analysis mode Settings Profiles to be used for the analysis can be selected for ROIs This Profile is always used for the ROI even if another Profile is set in operation Run In the column Settings Profile an existing Profile can be selected for an ROI Moreover the item settings can be used in the context menu for one or several selected ROIs When a Profile is created selected it is set in the column Settings Profile automatically Moreover Amplicons PCR Primers Skipped parts and Skipped Sequences can be defined for each ROI Amplicons PCR Primers are added in the same section Primers have to be added as primer pairs amplicons excluding the primer sequences are then created automatically Note In case primer sequences are entered all reads detected with these primer sequences are joined to the ROI also background For cases with background it is recommended to define amplicons only e Skipped parts Skipped sequences can be defined in operation Sequence and are entered in the corresponding fields here automatically A context menu for one or several sel
90. s is listed on each tab as well The Profile default is present already The first two entries Variation and Overview are selected in column Show on all tabs Gene file and databases Therefore the following is displayed by default 1 entry Variation Reference IDs are shown in the Variation Mutation table column web Ref for detected mutations The database is listed in paranthesis behind the ID 2 entry Overview Positions with WebRefs DB entry are shown in the location overview and electropherogram highlighted grey Information about the WebRef is shown in a tooltip when moving over the gene reference sequence Moreover information for variations mutations present in the SNP databases can be shown in additional columns of the Variation Mutation table Therefore please have a look at the User Manual SeqNext Filter DB Here mutations can be filtered For filtering special internal filters JSI can be used as as well as imported databases ClinVitae COSMIC ClinVar 1000 Genomes dbSNP When a filter is applied all filtered mutations are listed on tab temp Filter not permanent tab is cleared when the order is left To get a permanent filter a filter profile can be saved and applied in e operation Sequence for selected ROIs RO s Location context menu editing gt filters operation Joining for the complete order context menu editing gt settings tab Filter operation Run for the complete order tab Filter
91. s only used in case no wildtype is called in both sequencing directions The wildtype is missing if it does not reach the Min 26 coverage With this setting the option only 4 is set automatically tab Trimming gt Remove Ends is not available any more Note In case the options Adaptor and remove bases are used adaptors are removed first tab BAM SAM the following options are available to decide which information is used from the bam file and what is calculated by SeqPilot Section none perform mapping alignment quality filtering and variation calling with SeqPilot mapping alignment qualtiy filtering and variation calling is done with SeqPilot The corresponding information from the bam file is not used e section mixed o utilize mapping perform alignment quality filtering and variation calling with SeqPilot alignment pairwise alignment to the ROI to find the exact position of deletions insertions and SNPs quality filtering and variation calling is done by SeqPilot The mapping information is used from the bam file o utilize mapping and alignment perform quality filtering and variation calling with SeqPilot quality filtering and variation calling is done by SeqPilot The mapping and alignment information is used from the bam file e section full utilize mapping and alignment skip quality filters perform variation calling with SeqPilot Only the variation calling is done by SeqPilot SeqPilot does not apply
92. sequencing o cut expand coding exons refers to the coding sequence from start to stop codon e g setting is 5 20 and 3 30 all ROIs are cut expanded to 20 bases before and 30 What s new SEQUENCE Pilot 4 2 0 21 bases after the exon In case the ROI includes a start codon stop codon it is cut lexpanded to 20 bases before the start codon and 30 bases after the stop codon In case non coding exons are present they are set up as ROls but set to only mapping in column Analysis Mode o ROI refers to the ROI sequence genomic position e g setting is 5 20 and 3 30 All ROls are expanded to 20 bases before and 30 bases after the ROI In case the setting is 5 20 and 3 30 the first 20 bases and the last 30 bases of the ROI are cut Note can be used for sheared data In case Manifest files are loaded which include pseudogenes sequences these are set up as ROIs with the Analysis Mode only mapping automatically In case an error is present in the enrichment file you will get the following message after Build is pressed Not all lines are imported correctely Please look into the log file You can check the log file using the new button Log It opens a dialogue showing the following information Line line in the enrichment file Log In case there is an error the chromosomal position in that line was not translated into an ROI An accordi
93. ss the bwa files can also be downloaded from our ftp server 1 Therefore go to ftp ftpsrv jsi medisys de 2 Use the following login Username SeqPilotData Password SeqPilot 3 Download the file Genome hg19 hg19IndexFiles exe Execute the file and follow the installation instructions as destination directory C SeqPilot GeneFiles Genome is proposed automatically in case this isn t the folder of your SeqPilot installation please change the link to the GeneFiles folder of your SeqPilot installation Note This has only to be done for installations with existing genomes For newly downloaded genomes from our homepage the bwa files are included already New column Analysis Mode in the ROI List ROIs that should only be used for mapping can be defined to filter background reads If only mapping is checked the ROI is not shown in operation Sequence The Analysis Mode can be changed for all or selected ROIs simulatenously with the context menu in the ROI List Use the entry Analysis Mode gt all selected gt blanc only mapping Note A third option is available in RO Groups master file There known pseudogene sequences from Pseudogenes org can be imported All read that are filtered because of these three options are listed in the file UnusedReads txt that locates in the SeqNResults folder of your SEQUENCE Pilot installation in the folder for the order The reads have the flag 4 and the name of the ROI in front
94. t can be entered In case a shortcut is entered it is shown on the tab Protocol operation Sequence in the column event instead of the complete event Modules This column can not be changed In case an event is not available for all modules the module name is listed here What s new SEQUENCE Pilot 4 2 0 4 Order Info Order Info gives a statistic overview about order states You can search for a Date Date range and Module s in the section Select Orders It is then shown how many orders were loaded archived or extracted for the selected date range and module For active orders the number of complete incomplete technical and medical validated orders is shown Task Scheduler only for Client Server installations With the Task Scheduler you can set time intervals to automatically archive and or extract your orders regularily The tabs Archive and Extract are available to specify the time intervals and orders that should be archived extracted Note This feature can only be used if the User Right edit Scheduler is active For further information please have a look at the User Manual 1 5 Menu Help There are several new items available which explain possible Warnings for SeqNext HLA and or SeqNext e SeqNext MutationWarnings Warnings that occur in column Mutations of the sections Files ROI Groups Genes Chromosomes and ROls Locations SeqNext SeqNext HLA CoverageWarnings Warnings that occur in column Coverage in
95. tem show gt unused reads in section ROIs Note The Run ID can be found in the dialogue show Info operation Joining context menu of the Lower table or operation Sequence context menu of section File behind the entry ID 6 4 smMIP Processing Method In smMIP single molecule Molecular Inversion Probes a molecular tag is added to one of the reads of a read pair to be able to assign them to an individual capture event If a tag occurs more than once for a specific library amplicon either a consensus can be built to remove random sequencing errors or only one of the reads is considered for the read depth coverage within this amplicon Thus it is possible to detect low frequency or subclonal variation Method in SeqPilot a When starting a run in operation Run smMIP processing has to be enabled via three variables within the settings 1 On tab Trimming the length of the molecular tag e g 10 must be given in the field Remove Bases 5 Remove Bases 3 must stay empty On tab Expert Settings the boxes Unique reads only and Compl reads only must be checked 3 On tab Expert Settings the boxes Ignore paired end info and Allow unique paired end reads must not be checked Given this configuration a note should occur on tab Expert Settings smMIP processing enabled with tag length 10 Note Exactely two files have to be loaded in operation Run R1 and R2 files The R2 file the file with the reads including the molec
96. tended area a maximum number of seven bases is shown Basecalling The basecalling for positions with different bases called in forward and reverse direction changed Example reference base is C forward base is C and reverse base is T If the setting per dir is used no mutation can be called because the T was not found in forward direction No wildtype can be called because the C was not found in reverse direction For those positions no combined sequence is called In the Location overview positions where no combined sequence can be called are greyed out What s new SEQUENCE Pilot 4 2 0 37 6 12 9 Show New renmamed check jumpers are available required moves to positions that are ignored because they do not fullfill the Required Coverage settings operation Run tab Settings coverage here you can jump through positions in the selected RO Location sorted by coverage The position with the lowest coverage is jumped to first web mut was renamed in web mut Ref low was renamed in expected low quality was renamed in quality 6 12 10 Report In the report the different tabs of the Variation Mutation table can be printed separated now In the Print Preview the new table Variation is present to select which tab should be printed You can select several tabs the tabs will be printed as separated tables With Save as default your settings are saved In the dialogue Print Preview all loaded files for a run are shown in
97. tion Run and in operation ROI master file 5 3 Operation Run gz files Illumina can be loaded now without unzipping Loading of files Button For Client Server installations several files can be selected simultaneously now 6 Module SeqNext 6 1 Genomes On our homepage we offer hg38 for installation Therefore go to http www jsi medisys de genomes snp dbs 6 2 Import of Variation DB files What s new SEQUENCE Pilot 4 2 0 15 An installation package including the following databases is available for genome hg19 dbSNP Short Genetic Variations http www ncbi nlm nih gov SNP 1000 Genomes Catalog of Human Genetic Variation http www 1000genomes org ClinVar Sequence Variation and its relationship to human health http www ncbi nlm nih gov clinvar CLINVITAE Clinically observed genetic variants http clinvitae invitae com COSMIC Catalogue of somatic mutations in cancer http cancer sanger ac uk cancergenome projects cosmic These can be used to show known SNP information and for filter options see operation Sequence Variation Mutation table For the genomes available on our homepage we offer an exe file for easy installation of all Variation databases There are two installation packages we offer for download one referring to hg19 the other one referring to hg38 please select the correct one for your installation depends on the installed genome hg19 or hg38 there is
98. tion Sequence You can jump to positions with low quality using the jumper quality in the section Show Tab Trimming settings to trim adaptors or to automatically remove sequences at the ends of the reads can be entered Note In case the options Adaptor and remove bases are used adaptors are removed first o Adaptor Here adaptor sequences can be entered to trim or discard reads In your analysis you can get an overview about trimmed reads in section Files context menu show gt nfo The sequence entered here is searched for also reverse complement in all reads Enter an adapter sequence in 5 gt 3 direction The following fields can be edited for each entered adapter Position auto It is decided automatically if the adaptor locates at the 5 or 3 end 5 Adaptor can be found at the 5 end only 3 Adaptor can be found at the 3 end only Error rate here a percentage value can be entered as error rate wrong bases that the adapter can contain 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 o Remove bases Removes bases at the beginning and or end of each read Adaptors are removed prior to that Tab Expert settings o Basecalling Unique reads only If checked the coverage of identical reads is set to 1 o Read Processing Alignment evaluation e Skip evaluation I
99. to another allele then the reverse read e Tab Quality Score Here settings to exclude bases with bad quality from analysis can be set o Quality Score Threshold n case there is a value entered e g 15 only bases with a Quality Score above this value are counted to the coverage Positions with bad quality are greyed out and not used for analysis The Quality Score filter can be switched off by selecting Quality Score threshold off In 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 not called due to bad quality is shown in a lighter grey o Ignore Reads Threshold If the bases with bad quality in the complete read including primers adapters exceeds this value the read is ignored Bad quality means that the quality is below or equal the Quality Score Threshold setting or that Ns were called by the sequencer o 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 an ROI the warning quality is shown in the column What s new SEQUENCE Pilot 4 2 0 47 Coverage of section ROIs and Locations opera
100. uffix 2 Source Here you can search for ROIs with a certain Source The following sources are possible gene file genome chromosome range and fasta file The column Active can be changed for all or selected ROIs simultaneously with the context menu in the ROI List Use Active gt set remove gt all selected The field column Category was renamed into Panel The new column CNV probe type lists the CNV probe type For control ROIs this entry can be set to Control blanc means Target ROI An ROI including all information such as Skipped parts Skipped Sequences Homologies only mapping and Amplicons PCR Primer can be copied Use the context menu in the RO List and press copy The dialogue New Panel Suffix opens where you can select a Panel and a Suffix added to the ROI Name The section gnored Parts was renamed into Skipped Parts The section gnored Sequences Pseudogenes was renamed into Skipped Sequences Section Amplicons PCR Primers new column Multiplex No here optionally a PCR Multiplex number can be entered for each amplicon In section Sequence the chromosomal location the length of the sequence and the strand is displayed in front of the ROI sequence In case an ROI was defined wrong so that the location is not the defined exon anymore you will get the warning WARNING incorrect ROI E g BRCA1 E2 20 2000 is defind but exon 2 length is only 99 bp so the defined ROI is not BRCA1 E2 Button Import
101. ular tag has to be the second one listed in section Files operation Run b Processing 1 Reads are mapped to ROls aligned to their respective reference sequences and then assigned to amplicons within the ROIs What s new SEQUENCE Pilot 4 2 0 17 2 Single reads their paired read is missing i e has not been mapped to the same ROI or amplicon or does not fullfill the quality filters are discarded The discarded reads are added to the number of ignored reads see Error code 55 in the context of Unused Reads 3 The molecular tag is identified within the read pair and all duplicate read pairs with respect to an amplicon are discarded keeping those base sequence which occurred the most within the group of duplicates These reads are added to the number of duplicate reads see Error code 58 in the context of Unused Reads c Summary window gt tab Amplicon An additional column Dupl Reads has been added on tab Amplicon showing the amount of reads that have been discarded in processing step 3 The absolute value is followed by a percentage indicating the amount of duplicates within the set of complete reads which is the sum of Aligned Reads and Dupl Reads 6 5 Menu Help New item UnusedReadsErrorCodes You can open a list of all possible error codes for unused reads present in UnusedReads txt and operation Sequence sections Files and ROls Locations context menu item show gt unused reads 6 6 Operation ROI
102. used for the current analysis are shown ROIs can 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 Skipped Parts or Sequences are highlighted After doing a recalculate the highlighting disappears and the new ROI information are also shown on tab Current analysis o Info n the section General the number of assigned and aligned reads for the selected ROI are shown 9 5 2 Location overview A location overview is available above the electropherogram It gives an overview of the selected location in the dialogue part Locations You can jump to a location within the electropherogram by clicking on the overview Moreover in case several amplicons are defined for the ROI they are shown as red lines below the location overview The coverage of the location is shown graphically below the location overview You can jump to the positions by clicking on the graph The graph color changes from grey to pink if the coverage is below the Expected Coverage The Expected Coverage can be set in the Settings of the operation Run for each run In case there a bases with a coverage below there is also the hint expected in section ROls and Locations Moreover the coverage of bases that were not called due to bad quality is shown in a lighter grey 9 5 3 Combined HT1 and HT2 sequence There is several new information available
103. value compared to the first base This value is very important and needs to be adapted according to your data Example Basecalling coverage 20 96 o Coverage A 100 C 20 Allele 1 A is set Allele 2 C is set o Coverage A 100 C 19 Allele 1 A is set Allele 2 A is set C is regarded as background and not called 2 Required Coverage If the settings are not fulfilled the positions are greyed out and not used for result calculation Min abs coverage Minimum absolute coverage at each position You can choose if the value has to be reached in both sequencing directions together select combined or in forward and reverse separated select per dir Ratio read directions This is the radio between the forward and reverse coverage 3 Expected Coverage warning Min abs coverage The value entered here is shown as a red dotted line in the electropherogram coverage overview In case there are positions with a coverage below this value you get a warning There is the hint expected in the column Coverage of sections RO s and Locations and the graph color of the coverage graph changes from grey to pink o Warning Warnings are shown in operation Sequence in the column Warning of the Files Genes ROIs and Locations table Min reads per Allele Warning F in case the number of reads joined to allele 1 or 2 is below this value Two options can be additionally set for this setting perfect matches only Warning F in case the
104. y to the reference and starting from a certain position the bases can not be aligned anymore e g as expected for transversion translocations The position where the not aligned part of the read starts is listed in the table In the sequences the not aligned part is cut off there is a red arrowhead present showing in the direction of the cut off To see the complete reads click on the arrowhead and select show mutation A warning is only shown in case the frequency of the sequences that can not be aligned reaches the Warning value defined in the Settings operation Run Expert Setting Warning default 50 o possible del possible ins This warning is shown in case there are possible deletions insertions in the ROI that were not called The warning is present in case there What s new SEQUENCE Pilot 4 2 0 34 are different overlapping insertion deletions present for at least one position of the ROI Together the coverage of these insertions deletions at a position must fullfill the Settings Profile selected in the operation Run o fragment size possible deletion insertion Only present for sheared paired end data without amplicons e g Haloplex data The setting randomly sheared has to be set in the Settings operation Run For these data the fragment size is checked This gives a hint for deletions insertions Too short fragments give a hint for an insertion too long fragments a hint for a deletion The fragment size can be checked usin
105. y mutations with the entered Mut Effect are listed Frequency Values can be entered to search for mutations that are equal above or below a certain Frequency The values are entered with gt or lt or no sign equal in front in the hierarchy homo hetero of total Examples o 5 search for homozygous mutations with a frequency gt 5 o 10 search for heterozygous mutations with a frequency equal 10 o of lt 20 search for mutations with a total number of archived orders lt 20 o gt 10 lt 5 of gt 30 search for mutations which have a frequency homozygous gt 10 heterozygous 5 and a total number of archived orders 30 o gt 20 of gt 100 search for mutations whith a sum of heterozygous and homomzygous frequency gt 20 and a total number of archived orders gt 100 o 10 of gt 40 search for mutations whith a sum of heterozygous and homomzygous frequency equal 10 and a total number of archived orders gt 40 Date List mutations with the selected Changed date only The Changed date is listed in the dialogue Mutation Date Range List mutations with the selected Changed date range only The Changed date is listed in the dialogue Mutation Type List mutations of a special type only C base change D deletion insertion ndel indel g cDNA Search for mutations found in a special Seq Type genomic or cDNA above or below a certain value The values are entered with gt or

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