Isaac Whole Genome Sequencing v2 User Guide - Support
Contents
1. joyM Deesi 22 Statistic Definition Insertion Het Hom ratio Ratio of the number of heterozygous to homozygous insertions Deletion Het Hom ratio Ratio of the number of heterozygous to homozygous deletions SmallVariantStatisticsFlag Flags whether SmallVariantStatistics was run 1 means that it was run SVStatisticsFlag Flags whether SVStatistics was run 1 means that it was run CNVStatisticsFlag Flags whether CNVStatistics was run 1 means that it was run Sample Summary Report The Sample Summary Report presents the same data as the Resequencing_summary csv but in an easier to read format for humans These files are located in the results folder For a description of the presented metrics see Resequencing_summary csv on page 20 15050954 Rev B Isaac Whole Genome Sequencing Methods This chapter describes the methods that are used in the Isaac Whole Genome Sequencing app Isaac Aligner The Isaac aligner aligns DNA sequencing data single or paired end with read lengths and low error rates using the following steps Candidate mapping positions Identifies the complete set of relevant candidate mapping positions using a 32 mer seed based search Mapping selection Selects the best mapping among all candidates Alignment score Determines alignment scores for the selected candidates based on a Bayesian model Alignment output Generates final output in a sorted duplicate marked BAM file and summary file 12. INFO Additional information INFO fields are encoded as a semicolon separated series of short keys with optional values in the format lt key gt lt data gt data gVCF files use the following values END End position of the region described in this record BLOCKAVG min30p3a Non variant site block All sites in a block are constrained to be non variant have the same filter value and have all sample values in range x y y lt max x 3 x 1 3 All printed site block sample values are the minimum observed in the region spanned by the block SNVSB SNV site strand bias SNVHPOL SNV contextual homopolymer length CIGAR CIGAR alignment for each alternate indel allele RU Smallest repeating sequence unit extended or contracted in the indel allele relative to the reference If longer than 20 bases RUs are not reported REFREP Number of times RU is repeated in reference IDREP Number of times RU is repeated in indel allele FORMAT Format of the sample field FORMAT specifies the data types and order of the subfields gVCF files use the following values GT Genotype GQ Genotype Quality GQX Minimum of Genotype quality assuming variant position Genotype quality assuming non variant position DP Filtered base call depth used for site genotyping DPF Base calls filtered from input before site genotyping AD Allelic depths for the ref and alt alleles in the order listed For indels this value only includes reads
3. 1 Type Integer Description End position of the variant described in this record gt CHROM POS ID REF ALT QUAL FILTER INFO FORMAT NA19238chr1 51845 A PASS END 51862 Any field provided for a block of sites such as read depth using the DP key shows the minimum value that is observed among all sites encompassed by the block Each sample value shown for the block such as the depth DP is restricted to a range where Isaac Whole Genome Sequencing User Guide 1 Fi nding bulduanbasg awouan joyM Deesi 18 the maximum value is within 30 or 3 of the minimum For example for sample value range x y y lt xtmax 3 x 0 3 This range restriction applies to each of the sample values printed in the final block record Indel Regions Sites that are filled in inside of deletions have additional changes All deletions Sites inside of any deletion are marked with the deletion filters in addition to any filters that have already been applied to the site Sites inside of deletions cannot have a genotype or alternate allele quality score higher than the corresponding value from the enclosing indel Heterozygous deletions Sites inside of heterozygous deletions are altered to have haploid genotype entries e g 0 instead of 0 0 1 instead of 1 1 Heterozygous SNV calls inside of heterozygous deletions are marked with the SiteConflict filter and their genotype is unchanged Homozygous deletion
4. Come Raczy Roman Petrovski Christopher T Saunders Ilya Chorny Semyon Kruglyak Elliott H Margulies Han Yu Chuang Morten K llberg Swathi A Kumar Arnold Liao Kristina M Little Michael P Str mberg and Stephen W Tanner 2013 Isaac Ultra fast whole genome secondary analysis on Illumina sequencing platforms Bioinformatics 29 16 2041 3 bioinformatics oxfordjournals org content 29 16 2041 Candidate Mapping To align reads the Isaac aligner first identifies a small but complete set of relevant candidate mapping positions The Isaac aligner begins with a seed based search using 32 mers as seeds After the initial single seed search Isaac performs a multi seed search for only those reads that were not mapped unambiguously with a single seed Mapping Selection Following a seed based search the Isaac aligner selects the best mapping among all the candidates For paired end data sets all mappings where only one end is aligned called orphan mappings trigger a local search to find additional mapping candidates These candidates called shadow mappings are defined through the expected minimum and maximum insert size After optional trimming of low quality 3 ends and adapter sequences the possible mapping positions of each fragment are compared This step takes into account pair end information when available possible gaps using a banded Smith Waterman gap aligner and possible shadows The selection is based on the Smith Waterman s
5. Illumina Technical Support Table 5 Illumina General Contact Information Illumina Website www illumina com Email techsupport illumina com Table 6 Illumina Customer Support Telephone Numbers Region Contact Number Region Contact Number North America 1 800 809 4566 Italy 800 874909 Austria 0800 296575 Netherlands 0800 0223859 Belgium 0800 81102 Norway 800 16836 Denmark 80882346 Spain 900 812168 Finland 0800 918363 Sweden 020790181 France 0800 911850 Switzerland 0800 563118 Germany 0800 180 8994 United Kingdom 0800 917 0041 Ireland 1 800 812949 Other countries 44 1799 534000 Safety Data Sheets Safety data sheets SDSs are available on the Illumina website at support illumina com sds html Product Documentation Product documentation in PDF is available for download from the Illumina website Go to support illumina com select a product then click Documentation amp Literature Isaac Whole Genome Sequencing User Guide 3JUB SISSY B2ILY2A Ilumina San Diego Califomia 92122 U S A 1 800 809 ILMN 4566 1 858 202 4566 outside North America techsupport illumina com Www illumina com
6. dbSNP variant it is encouraged to use the rs number No identifier is present in more than one data record If there is no identifier available then the missing value is used REF Reference bases A C G T N there can be multiple bases The value in the POS field refers to the position of the first base in the string For simple insertions and deletions in which either the REF or one of the ALT alleles would otherwise be null empty the REF and ALT strings include the base before the event This modification is reflected in the POS field The exception is when the event occurs at position 1 on the contig in which case they include the base after the event If any of the ALT alleles is a symbolic allele an angle bracketed ID String lt ID gt the padding base is required In that case POS denotes the coordinate of the base preceding the polymorphism ALT Comma separated list of alternate non reference alleles called on at least one of the samples Options are Base strings made up of the bases A C G T N Angle bracketed ID String lt ID gt Break end replacement string as described in the section on break ends If there are no alternative alleles then the missing value is used QUAL Phred scaled quality score for the assertion made in ALT i e 10log_10 probability call in ALT is wrong If ALT is no variant this score is 10log 10 p variant If ALT is not this score is 10log_10 p no variant High QUAL score
7. length is calculated based on the locations at which a read pair aligns to the reference The read mapping information is parsed from the BAM files Minimum length of the sequenced fragment Maximum length of the sequenced fragment Standard deviation of the sequenced fragment length Total number of reads passing filter for this sample Percentage of reads passing filter that aligned The percentage of bases with a quality score of 30 or higher The average percentage of mismatches across both reads 1 and 2 over all cycles Total number of Single Nucleotide Variants present in the data set passing the quality filters SNVs passing variants filter 100 Number of SNVs in dbSNP Number of SNVs Transition rate of SNVs that pass the quality filters divided by transversion rate of SNVs that pass the quality filters Transitions are interchanges of purines A G or of pyrimidines C T Transversions are interchanges of purine and pyrimidine bases for example A to T Number of heterozygous SNVs Number of homozygous SNVs Total number of indels present in the data set passing the quality filters Insertions passing variant filters Deletions passing variant filters 100 Number of Indels in dbSNP Number of Indels 100 Number of insertions in dbSNP Number of insertions 100 Number of deletions in dbSNP Number of deletions Number of heterozygous indels Number of homozygous indels 21 nding bulduanbasg awouan
8. summarization by the SNP caller SNP Calling The variant caller runs a series of filters on the set of filtered and realigned reads for SNP calling without affecting indel calls First any contiguous trailing sequence of N base calls is trimmed from the ends of reads Using a mismatch density filter reads having 15050954 Rev B an unexpectedly high number of disagreements with the reference are masked as follows The variant caller treats each insertion or deletion as a single mismatch Base calls with more than two mismatches to the reference sequence within 20 bases of the call are ignored If the call occurs within the first or last 20 bases of a read the mismatch limit is applied to a 41 base window at the corresponding end of the read The mismatch limit is applied to the entire read when the read length is 41 or shorter Indel Genotypes The variant caller filters out all bases marked by the mismatch density filter and any N base calls that remain after the end trimming step These filtered base calls are not used for site genotyping but appear in the filtered base call counts in the variant caller output for each site All remaining base calls are used for site genotyping The genotyping method heuristically adjusts the joint error probability that is calculated from multiple observations of the same allele on each strand of the genome This correction accounts for the possibility of error dependencies This method treats
9. variant Variants are ordered by position ID The rs number for the SNP obtained from dbSNP If there are multiple rs numbers at this location the list is semi colon delimited If no dbSNP entry exists at this position the missing value is used Isaac Whole Genome Sequencing User Guide 1 3 nding bulduanbasg awouan joyM Deesi 14 Setting REF ALT QUAL FILTER FORMAT INFO Description The reference genotype For example a deletion of a single T can be represented as reference TT and alternate T The alleles that differ from the reference read For example an insertion of a single T can be represented as reference A and alternate AT A Phred scaled quality score assigned by the variant caller Higher scores indicate higher confidence in the variant and lower probability of errors For a quality score of Q the estimated probability of an error is 10 Q 10 For example the set of Q30 calls has a 0 196 error rate Many variant callers assign quality scores based on their statistical models which are high relative to the error rate observed in practice See VCF FILTER Entries on page 16 for possible entries See VCF FORMAT Entries on page 16 for possible entries See VCF INFO Entries on page 16 for possible entries 15050954 Rev B Setting Description INFO Illumina Annotation Service IAS provided annotations are e CSOT Transcript consequence as predicted by Variant Effect Predictor ww
10. Data Analysis Started Alignment for Sample SampleName If SVICNV is selected Detect CNV for Sample SampleName If SVICNV is selected Detect SV for Sample SampleName Variant analysis for Sample SampleName Statistics evaluation for Sample SampleName Oo 20 N BD oO FPF Q N e Report generation for Sample SampleName m o Analysis Completed for Sample SampleName m m Finalizing Analysis Results for Sample SampleName N Finished Finalizing Analysis Results The Isaac Whole Genome Sequencing app provides an overview of the input samples and settings that were specified when setting up the Isaac Whole Genome Sequencing run Isaac Whole Genome Sequencing Files 12 The Files page provides access to the output files See the following pages for descriptions BAM Files on page 13 VCF Files on page 13 gVCF Files on page 17 Resequencing summary csv on page 20 Sample Summary Report on page 22 15050954 Rev B BAM Files The Sequence Alignment Map SAM format is a generic alignment format for storing read alignments against reference sequences supporting short and long reads up to 128 Mb produced by different sequencing platforms SAM is a text format file that is human readable The Binary Alignment Map BAM keeps the same information as SAM but in a compressed binary format that is only machine readable Detailed Description The file naming convention for aligned reads in BAM format is as f
11. Isaac Whole Genome Sequencing v2 Introduction Running Isaac Whole Genome Sequencing Isaac Whole Genome Sequencing Output Isaac Whole Genome Sequencing Methods 2 Technical Assistance O QD DM Ca LZS ae Ss Sa oces tO a ILLUMINA PROPRIETARY illumina 15050954 Rev B August 2014 This document and its contents are proprietary to Illumina Inc and its affiliates Illumina and are intended solely for the contractual use of its customer in connection with the use of the product s described herein and for no other purpose This document and its contents shall not be used or distributed for any other purpose and or otherwise communicated disclosed or reproduced in any way whatsoever without the prior written consent of Illumina Illumina does not convey any license under its patent trademark copyright or common law rights nor similar rights of any third parties by this document The instructions in this document must be strictly and explicitly followed by qualified and properly trained personnel in order to ensure the proper and safe use of the product s described herein All of the contents of this document must be fully read and understood prior to using such product s FAILURE TO COMPLETELY READ AND EXPLICITLY FOLLOW ALL OF THE INSTRUCTIONS CONTAINED HEREIN MAY RESULT IN DAMAGE TO THE PRODUCT S INJURY TO PERSONS INCLUDING TO USERS OR OTHERS AND DAMAGE TO OTHER PROPERTY ILLUMINA DOES NOT ASSUME ANY LIABILITY ARISING
12. Mb MG1655 Drosophila melanogaster 139 5 Mb Human 3 3 Gb Mus musculus 2 6 Gb PhiX Illumina 5386 b Rattus norvegicus 2 9 Gb Rhodobacter sphaeroides 4 6 Mb 2 4 1 Saccharomyces cerevisiae 12 2 Mb Staphylococcus aureus 12 8 Mb NCTC 8325 Isaac Whole Genome Sequencing User Guide Data Size 666 Mb 28 Gb 50 Mb 49 Mb 1 5 Gb 35 Gb 28 Gb 57 Kb 31 Gb 49 Mb 129 Mb 135 Mb Reads for 2 x 100 million 3 33 140 00 0 25 0 25 7 33 175 00 140 00 282 77 155 00 0 25 0 65 0 68 Reads for 2 x 150 million 2 22 93 33 0 17 0 16 4 88 116 67 93 33 188 51 103 33 0 16 0 43 0 45 Running Isaac Whole Genome Sequencing 1 Click the Apps button 2 Find Isaac Whole Genome Sequencing v2 in the list and click the Launch button 3 Read the End User License Agreement and permissions and click Accept if you agree 4 Fill out the required fields in the Isaac Whole Genome Sequencing input form a Analysis Name Provide the analysis name Default name is the app name with the date and time the app session was started Save Results To Select the project that stores the app results Sample Browse to the sample you want to analyze and select the checkbox Reference Genome Select the reference genome Enable SV CNV calling If selected structural variants SVs and copy number variants CNVs are called using the tools Grouper and CNVSeg respectively For more information see Large Indel and Stru
13. OUT OF THE IMPROPER USE OF THE PRODUCT S DESCRIBED HEREIN INCLUDING PARTS THEREOF OR SOFTWARE OR ANY USE OF SUCH PRODUCT S OUTSIDE THE SCOPE OF THE EXPRESS WRITTEN LICENSES OR PERMISSIONS GRANTED BY ILLUMINA IN CONNECTION WITH CUSTOMER S ACQUISITION OF SUCH PRODUCT S FOR RESEARCH USE ONLY 2014 Illumina Inc All rights reserved Illumina 24sure BaseSpace BeadArray BlueFish BlueFuse BlueGnome cBot CSPro CytoChip DesignStudio Epicentre GAIIx Genetic Energy Genome Analyzer GenomeStudio GoldenGate HiScan HiSeq HiSeq X Infinium iScan iSelect ForenSeq MiSeq MiSeqDx MiSeq FGx NeoPrep Nextera NextBio NextSeq Powered by Illumina SeqMonitor SureMDA TruGenome TruSeq TruSight Understand Your Genome UYG VeraCode verifi VeriSeq the pumpkin orange color and the streaming bases design are trademarks of Illumina Inc and or its affiliate s in the U S and or other countries All other names logos and other trademarks are the property of their respective owners Introduction After BaseSpace has generated FASTQ files containing the base calls and quality scores of a run you can use the Isaac Whole Genome Sequencing app to analyze the sequencing data The resulting data are analyzed in two steps alignment to the reference genome followed by assembly and variant calling The Isaac Whole Genome Sequencing app uses the following modules for analysis Alignment with the Isaac Alignment Software Variant calling wit
14. Service IAS can be accessed through a binary annotation ANT file that accompanies the VCF file Isaac Whole Genome Sequencing VCF Entries The VCF files for Isaac Whole Genome Sequencing can have the following entries in the FILTER Table 2 Entry FORMAT and INFO fields VCF FILTER Entries Description IndelConflict Locus is in region with conflicting indel calls SiteConflict Site genotype conflicts with proximal indel call typically a heterozygous SNV call made inside of a heterozygous deletion LowGQx Locus GOX is less than 30 or not present HighDPFRatio The fraction of base calls filtered out at a site is greater than 0 4 HighSNVSB SNV strand bias value SNVSB exceeds 10 HighDepth Locus depth is greater than 3x the mean chromosome depth Table 3 VCF FORMAT Entries Entry Description GOX Minimum of Genotype quality assuming variant position Genotype quality assuming non variant position GT Genotype GQ Genotype Quality DP Filtered base call depth used for site genotyping DPF Base calls filtered from input before site genotyping AD Allelic depths for the ref and alt alleles in the order listed For indels this value only includes reads that confidently support each allele posterior probability 0 999 or higher that read contains indicated allele vs all other intersecting indel alleles DPI Read depth associated with indel taken from the position preceding the indel Table 4 VCF INFO Entries Entry Descrip
15. argeted sequencing gVCF is also an appropriate choice to represent and compress the results gVCE is a text file format stored as a gzip compressed file genome vcf gz Compression is further achieved by joining contiguous non variant regions with similar properties into single block VCF records To maximize the utility of gVCF especially for high stringency applications the properties of the compressed blocks are conservative Block properties like depth and genotype quality reflect the minimum of any site in the block The gVCF file can be indexed creating a tbi file and used with existing VCF tools such as tabix and IGV making it convenient both for direct interpretation and as a starting point for tertiary analysis For more information see sites google com site gvcftools home about gvcf The following conventions are used in the variant caller gVCF files Samples per File There is only one sample per gVCF file Non Variant Blocks Using END Key Contiguous non variant segments of the genome can be represented as single records in gVCF These records use the standard END INFO key to indicate the extent of the record Even though the record can span multiple bases only the first base is provided in the REF field to reduce file size The following is a simplified segment of a gVCF file describing a segment of non variant calls starting with an A on chromosome 1 from position 51845 to 51862 INFO lt ID END Number
16. assifies read pairs as either InsertionPair DeletionPair InversionPair TandemDuplicationPair or ChimericPair according to which type of structural variant an anomalously mapped read pair is associated Isaac Whole Genome Sequencing User Guide 2 D spoy 8 1 Bulsuenbag swouak joyM oees ClusterFinder Clusters reads based on their type and the position of their alignment Only reads of the same type are clustered together at this stage except shadow and semi aligned reads which can be clustered together ClusterMerger Associates clusters of various anomalous read types with shadow semi aligned read clusters which breakpoints can cause A breakpoint is a pair of bases that are adjacent in the sample genome but not in the reference Two clusters are merged if they share the read or if they agree on the position and length of the structural variant This information is inferred from read alignment orientation and distance ReadBroker Interchromosomal translocations yield chimeric read pairs where one read aligns to one chromosome and its partner aligns to another Because Grouper examines each chromosome individually the ReadBroker step is performed to join the information from chimeric read pairs across chromosomes After ReadBroker SmallAssembler Assembles reads in clusters into contigs using a de Bruijn method and iteratively assembles reads into contigs until all reads in the cluster are assembled It also produces a file c
17. changes of purines A G or of pyrimidines C T Transversions are interchanges between purine and pyrimidine bases for example A to T Variants by Sequence Context Statistic Definition Number in Genes The number of variants that fall into a gene Number in Exons The number of variants that fall into an exon Number in Coding Regions The number of variants that fall into a coding region Number in UTR Regions The number of variants that fall into an untranslated region UTR Number in Splice Site The number of variants that fall into a splice site region Regions Number in Mature The number of variants that fall into a mature microRNA microRNA Isaac Whole Genome Sequencing User Guide nding bulduanbasg awouan joyM Deesi Variants by Consequence Statistic Definition Frameshifts The number of variants that cause a frameshift Non The number of variants that cause an amino acid change in a coding region synonymous Synonymous The number of variants that are within a coding region but do not cause an amino acid change Stop Gained The number of variants that cause an additional stop codon Stop Lost The number of variants that cause the loss of a stop codon Structural Variants Summary This table breaks structural variant output into the classes of variants called and reports the total number and their overlap with annotated genes All counts are based on PASS filter variants Variant Class Defin
18. core and on the log probability of each mapping Alignment Scores The alignment scores of each read pair are based on a Bayesian model where the probability of each mapping is inferred from the base qualities and the positions of the mismatches The final mapping quality is the alignment score truncated to 60 for scores above 60 and possibly corrected to known ambiguities in the reference as flagged in the seeds Following alignment reads are sorted Further analysis is performed to identify duplicates and optionally to realign indels Isaac Whole Genome Sequencing User Guide 2 3 spoy 8 1 Bulsuenbag swouak joyM Les Alignment Output After sorting the reads the Isaac aligner generates compressed binary alignment output files called BAM bam files using the following process Marking duplicates Detection of duplicates is based on the location and observed length of each fragment The Isaac aligner identifies and marks duplicates even when they appear on oversized fragments or chimeric fragments Optical duplicates are already filtered out during RTA processing Realigning indels The Isaac aligner tracks previously detected indels over a window large enough for the current read length and applies the known indels to all reads with mismatches Generating BAM files The first step in BAM file generation is creation of the BAM record which contains all required information except the name of the read The Isaac aligner
19. ctural Variant Calls on page 25 and CNV Variant Caller on page 26 f Annotation Choose which gene and transcript annotation reference database to use nan o 5 If desired fill out the advanced fields in the Isaac WGS input form a Min GOX for Variants Enter GOX for variants GOX is the minimum of the GQ genotype quality and QUAL low quality filter which makes it a conservative filter Default value is 30 the maximum recommended is 99 b Max Strand Bias for Variants Choose the maximum allowed strand bias for variant calling This option filters for reads in which the differences in allele frequencies for forward and reverse strand reads is too high Default is 10 c FlagPCRDuplicates If selected PCR duplicates are flagged in the BAM files and not used for variant calling PCR duplicates are defined as paired end reads generated from two clusters that have the exact same alignment positions for each read Optical duplicates are already filtered out during RTA processing Figure 2 Isaac Whole Genome Sequencing Input Form Analysis Name Isaac Whole Genome Sequencingv2 027 gt Continue Save Results To Select a Project o Sample Select Sample 0 Reference Genome Human UCSC hg19 Enable SV CNV Calling o Annotation RefSeq Ensembl o v Advanced Min GQX for Variants 3 Max Strand Bias for Variants 10 O Flag PCR Duplicates go 6 Click Continue 6 15050954 Rev B The Isaac Whole Genome Sequencin
20. g app now starts analyzing your sample When completed the status of the app session is automatically updated and you receive an email NOTE f If needed you can merge sampleF ASTQ files in BaseSpace See the BaseSpace User Guide for more information Bulouenbes awouak joyM Deesi Buluuny Isaac Whole Genome Sequencing User Guide T Isaac Whole Genome Sequencing Output This chapter describes the output that the Isaac Whole Genome Sequencing app produces To go to the results click the Projects button then the project then the analysis Figure 3 Isaac Whole Genome Sequencing Output Navigation Bar i Analysis Info Inputs E output Files hl Analysis Reports NA12878 51 App Result When the App Session is completed you can access your output through the left navigation bar which provides the following Analysis Info an overview of the app session settings See Analysis Info on page 11 for a description Inputs overview of input settings see Inputs on page 12 Output Files access to the output files organized by sample and app session See Isaac Whole Genome Sequencing Files on page 12 for descriptions Analysis Reports access to analysis metrics for each sample See Analysis Reports on page 8 for a description Analysis Reports The Isaac Whole Genome Sequencing app provides an overview of statistics per sample on the sample pages A brief description of the metrics is below Alignment Summary Statist
21. gy SO terms www ensembl org info genome variation predicted_ data html consequences and typically are either regulatory _ region variantorTF binding site variant e AF The allele frequency from all populations of 1000 genomes data e AA The inferred allele ancestral to the chimpanzee human lineage e GMAF Global minor allele frequency GMAF technically the frequency of the second most frequent allele Format GlobalMinor Allele AlleleFreqGlobalMinor e EVS Allele frequency sample count and coverage taken from the Exome Variant Server EVS Format AlleleFreqEVS EVSCoverage EVSSamples e cosmic The numeric identifier for the variant in the Catalogue of Somatic Mutations in Cancer COSMIC database cancer sanger ac uk cancergenome projects cosmic e clinvar Clinical significance from the ClinVar database www ncbi nlm nih gov clinvar e phastCons Denotes if the variant is an identical or similar sequence that occurs between species and maintained between species throughout evolution SAMPLE The sample column gives the values specified in the FORMAT column One MAXGT sample column is provided for the normal genotyping assuming the reference For reference a second column is provided for genotyping assuming the site is polymorphic Isaac Whole Genome Sequencing User Guide 1 D nding bulduanbasg awouan joyM Deesi 16 L NOTE The full set of provided annotations from the Illumina Annotation
22. h the Isaac Variant Caller Structural variant and large indels with the Large Indel and Structural Variant Caller Copy number variation CNV analysis with the CNV Variant Caller These modules produce the following output Realigned and duplicate marked reads in BAM file format Variants in VCF file format An additional Genome VCF gVCF file This file features an entry for every base in the reference which differentiates reference calls and no calls and a summary of quality In addition there is an annotation and metric generation step with the following output Summary pages A Resequencing summary csv file Annotated VCF file This binary file can be loaded into VariantStudio for viewing see www illumina comj clinical clinical_informatics illumina variantstudio ilmn See Isaac Whole Genome Sequencing Methods on page 23 and Isaac Whole Genome Sequencing Output on page 8 for more information Isaac Whole Genome Sequencing User Guide 3 UOI 2NP0 J U Figure 1 Isaac Whole Genome Sequencing Workflow Variant Calling Isaac Variant Caller Per Sample Reads FASTQ Alignment Isaac Alignments BAM Structural Variation CNV CNV Variant Caller Summary Pages Versions VCF gVCF Annotation Metric Generation Resequencing Summary csv Annotated VCF The following module versions are used in the Isaac Whole Genome Sequencing app Isaac 01 13 10 21 Isaac Variant Caller 2 0 17 CNV Var
23. iant Caller CNV seg 2 2 4 Structural Variant Caller Grouper 1 4 2 Samtools 0 1 18 Tabix 0 2 5 r1005 Current Limitations Before running the Isaac Whole Genome Sequencing app be aware of the following limitations 15050954 Rev B Currently the app does not support mate pair or other non forward reverse styles of paired end sequencing Currently the app does not support annotation of non human genomes Requires a minimum read length of 32 bp and a maximum read length of 150 bp Minimum recommended data set size is enough data to yield 10 x coverage after alignment of the genome being sequenced See Table 1 for recommended minimum number of reads for 10 x coverage Maximum data set size must be fewer than 200 Gigabases which equates to the following Approximately 1 billion reads assuming 2 x 100 Approximately 665 million reads assuming 2 x 150 Completedjobinfo xml may not print all statistics Sample name length has a maximum of 32 characters UOI 2NP0 J U GOX can be entered as any value although the maximum recommended value is 99 For recommended minimum number of reads for 10 x coverage for different species see Table 1 The number of reads listed yields 10 x coverage with an additional 596 to account for unaligned reads Table 1 Recommended Minimums for 10 x Coverage Genome Size Arabidopsis thaliana 63 4 Mb Bos taurus 2 65 Gb Escherichia coli K 12 4 7 Mb DH10B Escherichia coli K 12 4 6
24. ic Definition Number of Total number of reads passing filter for this sample reads Coverage Total number of aligned bases divided by the genome size Percent Percentage of paired reads that have duplicates Duplicate Paired Reads Fragment Median length of the sequenced fragment The fragment length is calculated Length based on the locations at which a read pair aligns to the reference The read Median mapping information is parsed from the BAM files 8 15050954 Rev B Statistic Fragment Length Standard Deviation Definition Standard deviation of the sequenced fragment length Read Statistics Statistic Percent Aligned Percent Q30 Mismatch Rate Definition Percentage of reads passing filter that aligned The percentage of bases with a quality score of 30 or higher The average percentage of mismatches across both reads 1 and 2 over all cycles Small Variants Summary This table provides metrics about the number of SNVs insertions and deletions Statistic Total Passing Percent found in dbSNP Het Hom Ratio Ts Tv Ratio Definition Total number of variants present in the data set that pass the variant quality filters 100 Number of variants in dbSNP Number of variants Number of heterozygous variants Number of homozygous variants Transition rate of SNVs that pass the quality filters divided by transversion rate of SNVs that pass the quality filters Transitions are inter
25. ition CNV Number of copy number variations Insertions Number of insertions Tandem duplications Number of tandem duplications Deletions Number of deletions Inversion Number of inversions Coverage Histogram The coverage histogram shows the number of reference bases plotted against the depth of coverage read depth It has the following features The dropdown menu allows you to look at the overall picture or highlight a particular chromosome The Fix Y Scale checkbox allows you to keep the Y Scale the same when comparing multiple chromosomes The Export TSV link allows you to export the coverage data in a tab separated TXT file 1 O 15050954 Rev B Figure 4 Isaac Whole Genome Sequencing Coverage Histogram Coverage Histogram Overall 7 Fix Y Scale A Export TSV d il w w a a Read Depth of Reference Bases o 20 Analysis Info This app provides an overview of the analysis on the Analysis Info page A brief description of the metrics is below Row Definition Name Name of the app session Application App that generated this analysis Date started Date and time the app session started Date completed Date and time the app session completed Duration Duration of analysis Session Type The number of nodes used Size Total size of all output files Status Status of the app session Log Files Clicking the Log Files link at the b
26. lization by deriving a ratio between a test and the reference Window size is fixed by default to 100 bp The HMM model with Gaussian emission distribution is used for segmentation A bin exclusion criterion less than 10 of build coverage in both samples is applied 15050954 Rev B The reference for CNV normalization is an alignability measure that is meant to gauge the probability of a position aligning to a single unique region of the genome In detail the notion of alignability for reads of length k is as follows given a map M that for a fixed read length k and any position P in a genome G stores at M P the number of occurrences in G of the k mer that starts at P for a given position P in G define the overlap set of P as the k mers that overlap P The alignability of P is the proportion of this overlap set that is unique Variant Scoring After copy number assignment each CNV call is assigned a quality score based on a two sample t test Each counts ratio in a 1 kb window on each size of a breakpoint or half the length of a variant call whichever is smaller is compared using t test This test is based on the null hypothesis that there is no difference in coverage on each size of the breakpoint Obtained p values are then reported as Q scores on a Phred scale as 10 log10 Isaac Whole Genome Sequencing User Guide PP T spoy 8 1 Bulsuenbag swouak joyM oees Notes Technical Assistance For technical assistance contact
27. ollows SampleName_S bam where is the sample number determined by ordering in the sample sheet Go to samtools sourceforge net 5AM1 pdf to see the exact SAM specification BWA adds some custom fields to the BAM output See bio bwa sourceforge net bwa shtml 4 for a description VCF Files VCF is a text file format that contains information about variants found at specific positions in a reference genome The file format consists of meta information lines a header line and then data lines Each data line contains information about a single variant More information is available here www 1000genomes org wiki Analysis V ariant 20Call 20Format vcf variant call format version 41 VCE File Format The file naming convention for VCF files is as follows SampleName S vcf where is the sample number determined by ordering in the sample sheet The header of the VCF file describes the tags used in the remainder of the file A description of the tags is also provided here and on www broadinstitute org gatk guide article id 1268 Setting Description CHROM The chromosome of the reference genome Chromosomes appear in the same order as the reference FASTA file generally karyotype order POS The 1 based position of this variant in the reference chromosome The convention for vcf files is that for SNPs this base is the reference base with the variant For indels or deletions this base is the reference base immediately before the
28. ontaining the reads that were used to assemble the contig with a realignment to the contig sequence SpanContigs Uses the presence of nearby anomalous read pairs to determine whether to extend the search range used by the subsequent AlignContig step from its default AlignContig Computes a dynamic programming alignment of a contig to a region of the reference genome merges full or partial duplicate calls of the same event into a single call VariantFilter Removes all structural variants that overlap with gaps identified in UCSC gaps The UCSC gaps file defines regions of the genome that have not been sequenced DeletionGenotyper Assigns a genotype to all deletions CNV Variant Caller CNV variant caller is designed to identify copy number variants CNVs in diploid genomes using Hidden Markov Models HMM or unbalanced Haar wavelets The method adopts a count based approach for CNV calling and comprises two main steps 26 1 Pre processing step during which read depth is computed at each position and then filtered based on CpG islands assembly gaps telomeric centromeric regions Either alignability tracks or coverage tracks obtained from a pool of reference sample are used to normalize the data Counts or count ratios are produced as an output Segmentation of read counts ratios using fixed or variable bin size and a copy number assignment Normalization A single sample or a pool of reference samples is used for norma
29. ottom of the Analysis Info page provides access to Isaac Whole Genome Sequencing app log files Log files are located in a folder in the Output Files section The key log files to help follow data processing and debugging are the following CompletedJobInfo xml Contains information about the completed job Logging zip Contains all detailed workflow log files for each step of the workflow SampleSheetUsed csv A copy of the sample sheet generated at the end of a run Isaac Whole Genome Sequencing User Guide 1 1 nding bulduanbasg sWwousH joyM Deesi Inputs WorkflowError txt Workflow standard error output contains errors messages created while running the workflow WorkflowLog txt Workflow standard output contains details about workflow steps command line calls with parameters timing and progress The following files contain additional information in case components such as mono do not work as expected monoErr txt Wrapper mono call error output contains anything that WorkflowError txt does not catch in most cases empty except one line monoOut txt Wrapper mono call standard output contains command calling the workflow and anything that WorkflowLog txt does not catch a NOTE For explanation about mono see www mono project com Isaac Whole Genome Sequencing Status The status of the Isaac Whole Genome Sequencing app session can have the following values Preparing Run Data Finished Preparing Run
30. reads data from base call BCL files that were written during primary analysis on the sequencer to generate the read names Data are then compressed into blocks of 64 kb or less to create the BAM file Isaac Variant Caller 24 The Isaac Variant Caller identifies single nucleotide polymorphisms SNPs and small indels using the following steps Read filtering Filters out reads failing quality checks Indel calling Identifies a set of possible indel candidates and realigns all reads overlapping the candidates using a multiple sequence aligner SNP calling Computes the probability of each possible genotype given the aligned read data and a prior distribution of variation in the genome Indel genotypes Calls indel genotypes and assigns probabilities Variant call output Generates output in a VCF file and a compressed genome variant call gVCF file See VCF Files on page 13 and gVCF Files on page 17 for details Indel Candidates Input reads are filtered by removing any of the following Reads that failed primary analysis quality checks Reads marked as PCR duplicates Paired end reads not marked as a proper pair Reads with a mapping quality less than 20 Indel Calling The variant caller proceeds with candidate indel discovery and generates alternate read alignments based on the candidate indels As part of the realignment process the variant caller selects a representative alignment to be used for site genotype calling and depth
31. s Homozygous reference and no call sites inside of homozygous deletions have genotype Sites inside of homozygous deletions that have a non reference genotype are marked with a SiteConflict filter and their genotype is unchanged wou Site and genotype quality are set to The described modifications reflect the notion that the site confidence is bound within the enclosing indel confidence On occasion the variant caller produces multiple overlapping indel calls that cannot be resolved into two haplotypes If this case all indels and sites in the region of the overlap are marked with the IndelConflict filter Genotype Quality for Variant and Non variant Sites The gVCF file uses an adapted version of genotype quality for variant and non variant site filtration This value is associated with the key GOX The GQX value is intended to represent the minimum of Phred genotype quality assuming the site is variant Phred genotype quality assuming the site is non variant The reason for using this value is to allow a single value to be used as the primary quality filter for both variant and non variant sites Filtering on this value corresponds to a conservative assumption appropriate for applications where reference genotype calls must be determined at the same stringency as variant genotypes i e An assertion that a site is homozygous reference at GOX gt 30 is made assuming the site is variant An assertion that a site i
32. s indicate high confidence calls Although traditionally people use integer Phred scores this field is permitted to be a floating point to enable higher resolution for low confidence calls if desired If unknown the missing value is specified Numeric FILTER PASS if this position has passed all filters i e a call is made at this position Otherwise if the site has not passed all filters a semicolon separated list of codes for filters that fail gVCF files use the following values PASS position has passed all filters IndelConflict Locus is in region with conflicting indel calls SiteConflict Site genotype conflicts with proximal indel call which is typically a heterozygous SNV call made inside of a heterozygous deletion Isaac Whole Genome Sequencing User Guide 1 Q nding bulduanbasg awouan joyM Deesi LowGQX Locus GOX minimum of Genotype quality assuming variant position Genotype quality assuming non variant position is less than 30 or not present HighDPFRatio The fraction of base calls filtered out at a site is greater than 0 3 HighSNVSB SNV strand bias value SNVSB exceeds 10 High strand bias indicates a potential high false positive rate for SNVs HighSNVHPOL SNV contextual homopolymer length SNVHPOL exceeds 6 HighREFREP Indel contains an allele that occurs in a homopolymer or dinucleotide track with a reference repeat greater than 8 HighDepth Locus depth is greater than 3x the mean chromosome depth
33. s a non reference genotype at GOX gt 30 is made assuming the site is non variant Section Descriptions The gVCF file contains the following sections Meta information lines start with and contain metadata config information and define the values that the INFO FILTER and FORMAT fields can have 15050954 Rev B The header line starts with and names the fields that the data lines use These fields are CHROM POS ID REF ALT QUAL FILTER INFO FORMAT followed by one or more sample columns Data lines that contain information about one or more positions in the genome If you extract the variant lines from a gVCF file you produce a conventional variant VCF file Field Descriptions The fixed fields CHROM POS ID REF ALT QUAL are defined in the VCF 4 1 standard provided by the 1000 Genomes Project The fields ID INFO FORMAT and sample are described in the meta information CHROM Chromosome an identifier from the reference genome or an angle bracketed ID String lt ID gt pointing to a contig POS Position The reference position with the first base having position 1 Positions are sorted numerically in increasing order within each reference sequence CHROM There can be multiple records with the same POS Telomeres are indicated by using positions 0 or N 1 where N is the length of the corresponding chromosome or contig ID Semi colon separated list of unique identifiers where available If this ID is a
34. that confidently support each allele posterior probability 0 999 or higher that read contains indicated allele vs all other intersecting indel alleles DPI Read depth associated with indel taken from the site preceding the indel SAMPLE Sample fields as defined by the header Resequencing summary csv The Isaac Whole Genome Sequencing app produces an overview of statistics for each sample in a comma separated values CSV format the resequencing summary csv The Resequencing summary csv presents the same data as the Sample Summary Report but in an easier to parse format These files are located in the results folder A brief description of the metrics is below 15050954 Rev B Statistic Sample ID Run Folder Fragment length median Fragment length min Fragment length max Fragment length SD Number of Reads Percent Aligned per read Percent Q30 per read MismatchRate per read SNVs All un NVs Passing Filters un NVs Percent found in bSNP Qu un NV Ts Tv ratio SNV Het Hom ratio Indels Insertions Passing Variants Deletions Passing Variants Indels Percent found in dbSNP Insertions Percent found in dbSNP Deletions Percent found in dbSNP Indel Het Hom ratio Isaac Whole Genome Sequencing User Guide Definition IDs of samples reported on in the file Run folders for samples reported on in the file Median length of the sequenced fragment The fragment
35. the highest quality base call from each allele and strand as an independent observation and leaves the associated base call quality scores unmodified Quality scores for subsequent base calls for each allele and strand are then adjusted This adjustment is done to increase the joint error probability of the given allele above the error expected from independent base call observations Variant Call Output After the site and indel genotyping methods are complete the variant caller applies a final set of heuristic filters to produce the final set of non filtered calls in the output The output in the genome variant call gVCF file captures the genotype at each position and the probability that the consensus call differs from reference This score is expressed as a Phred scaled quality score Large Indel and Structural Variant Calls The large indel and structural variant caller uses the series of modules described here and then generates output files in VCF 4 1 format Before ReadBroker StatsGenerator Computes summary statistics on insert sizes read orientation and alignment scores for each input BAM file AnomalousReadFinder Grouper processes chromosomes in chunks This method enables parallel execution and therefore faster performance AnomalousReadFinder examines all alignments in a block and classifies reads and read pairs as follows Classifies reads as either shadow unaligned or semi aligned partial or clipped alignment Cl
36. tion SNVSB SNV site strand bias SNVHPOL SNV contextual homopolymer length CIGAR CIGAR alignment for each alternate indel allele RU Smallest repeating sequence unit extended or contracted in the indel allele relative to the reference RUs longer than 20 bases are not reported REFREP Number of times RU is repeated in reference 15050954 Rev B Entry Description IDREP Number of times RU is repeated in indel allele END End position of the region described in this record BLOCKAVG Non variant site block All sites in a block are constrained to be min30p3a non variant have the same filter value and have all sample values in range x y y lt max x 3 x 1 3 All printed site block sample values are the minimum observed in the region spanned by the block gVCF Files This application also produces the Genome Variant Call Format file gV CF gVCF was developed to store sequencing information for both variant and non variant positions which is required for human clinical applications gVCF is a set of conventions applied to the standard variant call format VCF 4 1 as documented by the 1000 Genomes Project These conventions allow representation of genotype annotation and other information across all sites in the genome in a compact format Typical human whole genome sequencing results expressed in gVCF with annotation are less than 1 Gbyte or about 1 100 the size of the BAM file used for variant calling If you are performing t
37. w ensembl org info docs tools vep index htm version 72 Only canonical transcripts are included in the VCF file to maintain readability The ANT file contains consequences for all affected transcripts This binary file can be loaded into VariantStudio for viewing see www illumina com clinical clinical_informatics illumina variantstudio ilmn A comma separated list for each affected gene is provided Each entry in the list includes the HGNC gene symbol when available transcript ID and functional consequences in a delimited format HGNC TranscriptID Consequence If the annotation source selected was RefSeq then many of the TranscriptIDs begin with NM If the selected annotation source was Ensembl then the TranscriptIDs begin with ENST The consequences are indicated using valid Sequence Ontology SO terms www ensembl org info genome variation predicted_ data html consequences CSOR Regulatory consequence as predicted by Variant Effect Predictor www ensembl org info docs tools vep index htm version 72 A comma separated list for each affected regulatory region including transcription factor binding sites is provided using the following delimited format RegulatoryID Consequence The annotations provided in this field come from the Ensembl database of regulatory features even if RefSeq was selected as the annotation source Many of the RegulatoryIDs begin with ENSR The consequences are indicated using valid Sequence Ontolo
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