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Whole-Genome Sequencing Services User Guide - Support

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1. o Partitioning The Isaac CNV Caller partitioning implements an algorithm for identifying regions of the genome such that their average counts are statistically different than average counts of neighboring regions The implementation is a port of the circular binary segmentation CBS algorithm 3 8 Part 15040892 Rev D Calling The algorithm briefly considers each chromosome as a segment The algorithm assesses each segment and identifies the pair of bins for which the counts in the bins between them are maximally different than the counts of the rest of the bins The statistical significance of the maximal difference is assessed via permutation testing If the difference is statistically significant then the procedure is applied recursively to the 2 or 3 segments created by partitioning the current segment by the identified pair of points Input to the algorithm is the output generated by the Isaac CNV Caller cleaning algorithm Because of the computational complexity of the algorithm O N the problem is divided into subchromosome problems followed by merging in practice Heuristics are used to speed up the permutation testing The final module of the Isaac CNV Caller algorithm is to assign discrete copy numbers to each of the regions identified by the Isaac CNV Caller partitioner A Gaussian model is used as the default calling method In this case both the mean and standard deviation are estimated from the data for the di
2. Picard requires large amounts of memory Picard reads data sequentially line by line from the BAM file and stores the reads in memory until both pairs of each read have been read Memory is reset only when the reads are printed Every read that does not have adjacent or near adjacent pairs requires more memory Therefore sort large BAM files when memory is a limiting factor For additional information about Picard Tools see picard sourceforge net command lineoverview shtml Download Picard Tools at sourceforge net projects picard files picard tools SAMtools Sort 44 SAMtools sort ensures that paired reads are next to each other so you can save a significant amount of memory by using SAM tools to sort the BAM files by name before running Picard Sort the bam file by name and output to sorted by name bam samtools samtools 0 1 19 samtools sort n Q 4 m 1G Example bam Example sorted Bam Size 79G Wall Clock Time 3 hrs 5 min Optional Parameters 4 This option tells samtools to run 4 threads m 1G This option tells samtools to use 1Gb of memory per thread For additional information about SAMtools see samtools sourceforge net Part 15040892 Rev D Reads Extraction Using SAMtools Flags The Bam Sam format contains a bitwise flag column that contains a hexadecimal which defines the nature of that read SAMtools allows you to easily filter on reads based on this flag There are 12 types of s
3. f 12 a combination of flag 4 and flag 8 4 8 gt include only if a read is unmapped and the mate is unmapped command outputs read pairs with both pairs unmapped Ssamtools view h f 12 Example bam Whole Genome Sequencing Services User Guide A 5 OV4 alla NVE Appendix Ilumina FastTrack Services Annotation Pipeline The Illumina FastTrack Services Annotation Pipeline provides variant annotation for Single Nucleotide variants SNVs insertions and deletions indels All annotations are provided in the INFO field of Sample_Barcode vcf gz file and documented in the header Larger variants CNAs SVs are not annotated with the full pipeline The annotation database is queried for each of the small variants input to the pipeline Both positional and allelic annotations can be returned for a given variant After querying the annotation database novel variants variants for which no annotation exists are then processed with VEP If VEP does not return an annotation for the variant it will remain unannotated Annotation Database Sources 46 The following table includes sources for the annotation databases Table 29 List of Annotation Database Sources Source Version Variant Effect Predictor 72 1000 Genomes Allele Frequencies v3 Release 20110521 ClinVar 20130905 COSMIC 65 dbSNP 137 HGNC RefSeq Mapping Updated daily NHLBI Exome Variant Server v 0 0 20 ESP6500SI V2 phastCons N A Release Date 06 01 2013 04
4. Any VCF file following the gVCF convention combines information on variant calls SNVs and small indels with genotype and read depth information for all nonvariant positions in the reference Because this information is integrated into a single file distinguishing variant reference and no call states for any site of interest is straightforward The following subsections describe the general conventions followed in any gVCF file and provide information on the specific parameters and filters used in the Isaac workflow gVCF output dy NOTE gVCF conventions are written with the assumption that only one sample per file is being represented Interpretation gVCFs file can be interpreted as follows Fast interpretation As a discrete classification of the genome into variant reference and no call loci This classification is the simplest way to use the gVCF The Filter fields for the gVCF file have already been set to mark uncertain calls as filtered for both variant and nonvariant positions Simple analysis can be performed to look for all loci with a filter value of PASS and treat them as called Research interpretation As a statistical genome Additional fields such as genotype quality are provided for both variant and reference positions to allow the threshold Whole Genome Sequencing Services User Guide 31 49168 JOA Sousas Analysis Overview between called and uncalled sites to be varied These
5. Variant Statistics This table breaks down SNVs and indels into total counts in overlapping regions and annotated consequences Complex indels are split into deletions and insertions where appropriate Consequence types for overlapping transcripts are counted under the most severe transcript consequence according to the annotation nodey Aeuwuuns Structural Variants Summary This table breaks CNV and SV output into the classes of variants called Their total PASS count and the number of overlapping genes are based on the annotation pipeline see Illumina FastTrack Services Annotation Pipeline on page 46 Circos Plot of Genome Variations The Circos plot provides visualization of structural variation ploidy and structural variations reported in the genome variation files VCF The Circos plot displays genome variation data in tracks with chromosomes circularly arranged Following is an example legend Labels are described from inside the circle to the outside Lda t l 60 ts TS 80 01011 i 100 4 110 120 430 0101111 140 b A A 150 paea Y 160 Cp mo rena Whole Genome Sequencing Services User Guide 2 O Table 25 Circos Plot Legend Label From Inner Circle to Outer Circle A Structural variants Legend B Number indels per Mb Analysis Deliverables C Number of SNVs per Mb D Copy number variation E Karyotype Chromosome position F Chromosome
6. ii giving the other party exclusive control and authority over the defense and settlement of such claim or action iii not admitting infringement of any intellectual property right without prior written consent of the other party iv not entering into any settlement or compromise of any such claim or action without the other party s prior written consent and v providing reasonable assistance to the other party in the defense of the claim or action provided that the party reimburses the indemnified party for its reasonable out of pocket expenses incurred in providing such assistance Third Party Goods and Indemnification Illumina has no indemnification obligations with respect to any goods originating from a third party and supplied to Purchaser Third party goods are those that are labeled or branded with a third party s name Purchaser s indemnification rights if any with respect to third party goods shall be pursuant to the original manufacturer s or licensor s indemnity Upon written request Ilumina will attempt to pass through such indemnity if any to Purchaser Part 15040892 Rev D Part 4 15040892 15040892 15040892 15040892 Revision History Revision Date D June 2015 E July 2014 B July 2013 A April 2013 Description of Change Revised documentation to reflect changes in version 4 of the Ilumina FastTrack WGS pipeline e Renamed Manta and Canvas to Isaac Structural Variant Caller and Isaac Copy Numb
7. vcf format El Sample Barcode GenotypingReport txt Genotyping SNPs tab delimited report Variations El Sample Barcode CNV vcf gz Copy number calls 10 kb in vcf format El Sample Barcode Indels vcf gz Small Insertion Deletion calls 1bp 50 bp in vcf format El Sample Barcode SNPs vcf gz Single nucleotide polymorphism SNVs calls in vcf format El Sample Barcode SV vcf gz Large Structural Variation calls 51 bp 10 kb in vcf format Sample Barcode genome vcf gz Genome vcf file containing SNVs indels and reference covered regions El Sample Barcode vcf gz vcf file containing basic annotations and SNV and indel calls md5sum txt checksum file for confirming file consistency L NOTE All the vcf files that Ilumina provides are compressed and indexed using tabix For details about tabix see the tabix manual in SAMtools at samtools sourceforge net tabix shtm The tabix index shows up as an additional Sample Barcode TYPE vcf gz tbi file It can be used for fast retrieval of targeted regions in the associated vcf gz file L NOTE For some VCF files a binary format of the annotations and their indexes are contained in corresponding vcf ant and vcf ant idx files respectively If the vcf ant file is maintained in the same directory as its VCF file the annotation information can be visualized alongside the variant call information when imported to VariantStudio Whole Genom
8. 2 copies Isaac CNV Caller identifies regions of the sample genome that are not present or present either one time or more than 2 times in the genome Isaac CNV Caller scans the genome for regions having an unexpected number of short read alignments Regions with fewer than the expected number of alignments are classified as losses Regions having more than the expected number of alignments are classified as gains Isaac CNV Caller is appropriately applied to low depth cytogenetics experiments low depth single cell experiments or whole genome sequencing experiments Isaac CNV Caller is not appropriate for whole exome experiments cancer studies or any other experiment with the following conditions Most of the genome is not assumed to be diploid Reads are not distributed randomly across the diploid genome Workflow Binning Isaac CNV Caller can be conceptually divided into 4 processes Binning Counting alignments in genomic bins Cleaning Removal of systematic biases and outliers from the counts Partitioning Partitioning the counts into homogenous regions Calling Assigning a copy number to each homogenous region These processes are explained in subsequent sections The binning procedure creates genomic windows or bins across the genome and counts the number of observed alignments that fall into each bin The alignments are provided in the form of a BAM file Isaac CNV Caller binning keeps in memory a collection of
9. D Detected Variant Classes Isaac SV Caller is able to detect all variation classes that can be explained as novel DNA adjacencies in the genome Simple insertion deletion events can be detected down to a configurable minimum size cutoff defaulting to 51 All DNA adjacencies are classified into the following categories based on the break end pattern Deletions Insertions Inversions Tandem Duplications Interchromosomal Translocations Known Limitations Isaac SV Caller cannot detect the following variant types Nontandem repeats amplifications Large insertions The maximum detectable size corresponds to approximately the read pair fragment size but note that detection power falls off to impractical levels well before this size FastTrack WGS service reports called variants that are 50 10 kb in size Small inversions The limiting size is not tested but in theory detection falls off below 200 bases So called microinversions might be detected indirectly as combined insertion deletion variants More general repeat based limitations exist for all variant types Power to assemble variants to break end resolution falls to O as break end repeat length approaches the read size Power to detect any break end falls to nearly O as the break end repeat length approaches the fragment size The method cannot detect nontandem repeats While Isaac SV Caller classifies novel DNA adjacencies it does not infer the higher level constructs i
10. GTTAACCTTAAGAT TACTTGATCCACTGATTCAACGTACCGTAACGS GAATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACT TGATCCACTGATTCAACGTACCGTAAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACT Ey REN elt BURA OPC T te GATAACAGTAACACACT TCTGTTAACCTTIAAGAT TACI TGATCCACTGAI TCAACGTACCGTAACGAACGTATCAAIT TGAGACTAAATATTAACGTACCATTAAGAGCTACCGTCTTCTGTTAACCTTAAGATTACTT CTGATT LOLA sie Oily A oly SET UC AA q Gee TAACGAACGTATCAAT IGAGACTAAATAT TAACGTACCAT TAAGAGCT GTG ITAACOTIAAGATIAGI IGATOCACTGATT GAACGIACOG TAL L AR els I ao KN SA L OET AAA REA CTGTTAACCTT ET A VY L RT AACGACG CTAAATA CAT TAAGAGCTAC T TACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATAT TAACGTACCAI TAAGAGCTACCGTGCAAC GAAAAG AACAGTAACAC SATAACAGTAACACAC TTETATTAACG I TAAGAT TACT GATOCACTOA GAACGIAGOG TRAO GANGO IAI CAAT I GAGAG AAATAT TAACG TAGGATAAGAGCIACCATGTIGTGT TACT TANGATTACT TGATCUACT GA ICAA ACCA PARAG TAC as aL AACCTTAAGAT TACI TGATCCACT KA AACGAACGTATCAAT TGAGACTAAATATTAACGTACCATTAAGAGCTACCGTGCAACGACGAACTTCTGTT A AAGATTACTIGA GCTACCGTGCAACGAAAATAACCTIAAGATTACTTGATCCACTGATTCAACGTACTICTGTTAACCTIAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAGCTACCGTGCAACGACGAAAAGAATGA GAMAAGAATGATAACAGTAACAC C LICTOTTAACC I TAAGATTACI TGATOGACTGATTCAACGT CCOTAAAGATTAGITGATOCACTGAT IOAACGTACOGTAACGAACGTAI CAATTG GAC TAAATAT TAACGTACCAT TAAGAGCTAC PE vet v A ee ee ee ta a Ey eA Ra TO UA Vi AV AEP V V v P TA OA CATA CA LL ACCAT TAAGAGCTACCGTGCAACAGTAACACACTTCTGT TAACCTTAAGAT TACTTGATCCACTGATTCAACGTACCGTAACG
11. GenCall score This score is a quality metric assigned to every genotype called and generally indicates their reliability GC scores have a maximum of 1 and are calculated using information from the clustering of the samples Each SNP is evaluated based on the angle of the clusters dispersion of the clusters overlap between clusters and intensity Genotypes with lower GC scores are located furthest from the center of a cluster and have a lower reliability The internal process identifier The SNP identifier An rsID for dbSNP content Part 15040892 Rev D Variations The variations folder contains the variant call output in VCF 4 1 format for the sample Each variant file that Illumina provides is compressed and includes an index that was created using tabix for fast range based access This is a summary of the outputs for each sample See Introduction on page 24 for details The VCF files are annotated with the FastTrack Services Annotation Pipeline See Illumina Fast Track Services Annotation Pipeline on page 46 for details Sample Barcode CNV vcf gz The CNV file contains large copy number variants from 10 kb output from the Isaac CNV Caller The following fields are utilized in the VCF file Table 8 INFO Fields ID Description END End position of the variant described in this record SVTYPE Type of structural variant Table 9 ALT Fields ID Description CNV Copy number variable region Table 10 FORMAT Field ID D
12. Specifications ii improper handling installation maintenance or repair other than if performed by Illumina s personnel iii unauthorized alterations iv Force Majeure events or v use with a third party s good not provided by Illumina unless the Product s Documentation or Specifications expressly state such third party s good is for use with the Product d Procedure for Warranty Coverage In order to be eligible for repair or replacement under this warranty Purchaser must i promptly contact Ilumina s support department to report the non conformance ii cooperate with Ilumina in confirming or diagnosing the non conformance and iii return this Product transportation charges prepaid to Whole Genome Sequencing Services User Guide Illumina following Ilumina s instructions or if agreed by Ilumina and Purchaser grant Illumina s authorized repair personnel access to this Product in order to confirm the non conformance and make repairs Sole Remedy under Warranty Ilumina will at its option repair or replace non conforming Product that it confirms is covered by this warranty Repaired or replaced Consumables come with a 30 day warranty Hardware may be repaired or replaced with functionally equivalent reconditioned or new Hardware or components if only a component of Hardware is non conforming If the Hardware is replaced in its entirety the warranty period for the replacement is 90 days from the date of shipment or the remaining p
13. ade E AN 12 Summa ODOM zara nan 18 PAalNedN cre ess ate eagle II AR Oe 22 e Tann Ran LE S ousa REE 5 Si nn E iai H gt tr a A tit pos l u Whole Genome Seguencing Services User Guide 4 Z JSjdeuo Analysis Folder Structure Overview This section details the files and folder structure for the single whole genome deliverable Several folders are batched together at delivery but each sample follows the same underlying format The files and folders generated for the whole genome deliverable are all keyed off a unique sample identifier Sample Barcode Usually this unique identifier is the barcode associated with a sample in the lab eg LP6000001 DNA_A01 but can be a common sample identifier for reference samples eg NA12878 Analysis Deliverables 5 Part 15040892 Rev D Result Folder Structure Under each Sample folder you can find the following file structure that contains analysis results 7 Sample Barcode Assembly El Sample Barcode bam Archival bam file for sample El Sample_Barcode bam bai Index for bam file El Sample Barcode SummaryReport csv Summary report in csv format El Sample Barcode SummaryReport pdf Summary report in pdf format Genotyping 7 Sample Barcode idats Folder containing genotyping intensity data files for the sample idat files and genotyping sample sheet El Sample Barcode Genotyping vcf gz Genotyping SNPs mapped to reference in
14. consecutive genomic windows such that each bin contains the same bin size or number of unique 35 mers The number of observed alignments present within the boundary of each bin is then counted from the alignment BitArrays The GC content of each bin is also calculated The chromosome genomic start genomic stop observed counts and GC content in each bin are output to disk The Isaac CNV Caller cleaning comprises the following 3 procedures that remove outliers and systematic biases from the count data computed in Isaac CNV Caller 1 Single point outlier removal 2 Physical size outlier removal 3 GC content correction These procedures are performed on the bins produced during the Isaac CNV Caller binning process Part 15040892 Rev D Single Point Outlier Removal This step removes individual bins that represent extreme outliers These bins have counts that are very different from the counts present in upstream and downstream bins Two values a and b are defined as to be very different when their difference is greater than expected by chance assuming a and b come from the same underlying distribution These values use the Chi squared distribution as follows u 0 5a 0 5b x2 a u b Y p 1 A value of x2 greater than 6 635 which is the 99th percentile of the Chi squared distribution with 1 degree of freedom is considered very different If a bin count is very different from the count of both upstream and downstream ne
15. fields can also be used to apply more stringent criteria to a set of loci from an initial screen External Tools gVCF is written to the VCF 4 1 specifications so any tool that is compatible with the specification such as IGV and tabix can use the file However certain tools are not appropriate if they Apply algorithms to VCF files that make sense for only variants calls as opposed to variant and nonvariant regions in the full gVCF Are only computationally feasible for variant calls For these cases extract the variant calls from the full gVCF file Special Handling for Indel Conflicts Sites that are filled in inside deletions have additional treatment Heterozygous Deletions Sites inside heterozygous deletions have haploid genotype entries ie 0 instead of 0 0 1 instead of 1 1 Heterozygous SNVs are marked with the SiteConflict filter and their original genotype is left unchanged Sites inside heterozygous deletions cannot have a genotype quality score higher than the enclosing deletion genotype quality Homozygous Deletions Sites inside homozygous deletions have genotype set to period and site and genotype quality are also set to period All Deletions Sites inside any deletion are marked with the filters of the deletion and more filters can be added pertaining to the site itself These modifications reflect the idea that the enclosing indel confidence bounds the site confidence Indel Conflicts I
16. mis a beret aa ACCATTAAGAGCTACCGTGCAACTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGTGCAACGACGAACTTCTGTTAACCTTAAGATTACTIGA GCTACCGTGCAACGAAAATAACCTTAAGATTACT TGATCCACTGATTCAACGTACTTCTGTTAACCTTAAGATTACTTGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT T GAGACTAAGCTACCGT GCAACGACGAAAAGAAT GA GAAAAGAATGATAACAGTAACACACTTCTGT TAACCTTAAGAT TA Le Pe ANS AB A CCGTAAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTA o ET GATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGTGCAACGACGAAAAGAATGATAACAGTAACACACTICTGT ACCA TAAGAGCTACOGTGCAA CA GTAACACACI TETGTTAACC I TAAGATTACITGATO ACTGATTOAACGTACOGTAAC GAACOTAT AATTGA GACTAAATA TA OTAC CATTA GAGCIACC GI GC AACCACGANANGAATOATAL GATAACAGTAACACACI TOTGTTAACCI TAAGAI T LR OTROK CTAAATATT Cn a DR Alo eee ee a LAS ao GTACCGTAACGAACGTATCATTAAGATTACTIGATCCACTGATTCAACG GTAACGAACGTATCAAI GACTAAATAT TAACGTACCAT TAAGAGCTACCGTGCAACGACGAAAAGAATGATAACAGTAACACA CT TOTGTTAACOTI CITGATOCACTG I ICAACGI TAAGATACITGAI CGACTGATI CAACGIACCGTAACGAACGIAICAATI GAGCI TCTGITAAGOI TAAG TTACI IGATOCAC GAL CAC GTACOGTAACOAA CC TATGAAI GAGA CT G CHA CGACI GAAAAGAATGATAACAGTAACACACT TCTGTTAACCI TAAGATTACTTGATCCACTGAI TCAACGTACCGTAAAGAT TACT TGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAI TGAGACTAAATAT TAACGTACCAI TAAGAGCTAC 5 E Illumina 5200 Illumina Way San Diego California 92122 U S A 1 800 809 ILMN 4566 1 858 202 4566 outside Nor
17. number 21 Description The structural variants described in Sample_ Barcode SV vcf gz are plotted in the central portions of the Circos plot From inner to outer left to right in the legend e Red Links Translocation break ends e Dark Gray Tandem Duplications per Mb e Dark Blue Inversions per Mb e Grey Deletions per Mb e Blue Insertions per Mb The density of PASS indels reported in Sample_ Barcode SV vcf gz in 1 Mb windows The scale of Y axis in the histogram indicates the counts The density of PASS SNVs reported in Sample_ Barcode SV vcf gz 1 Mb windows arbitrarily scaled in a histogram with Y axis pointing inward The copy number variations from Sample_ Barcode CNA vcf gz file The scale of Y axis in the histogram indicates the called level e Orange bar loss of copy fewer than 2 copies e Blue bar gain of copy greater than 2 copies max of 5 The standard Circos ideogram defining the chromosome position identity and color of cytogenetic bands and the reference coordinates along the chromosome Chromosome number 1 2 22 X Y Part 15040892 Rev D Whole Genome Sequencing Services User Guide Data Integrity The md5sum txt file is provided as a means of checking the integrity of the sample files and folders Immediately after sample quality check the md5sums or compact digital fingerprint for every file in the directory tree are generated If media failures compromise da
18. site is greater than 0 3 HighSNVSB SNV strand bias value SNVSB exceeds 10 IndelConflict The locus is in a region with conflicting indel calls LowGOX Locus GOX is less than 30 or not present SiteConflict The site genotype conflicts with the proximal indel call This is typically a heterozygous SNV call made inside a heterozygous deletion Part 15040892 Rev D Summary Report The Sample Barcode SummaryReport pdf report contains an overview of the results for the sample In the report you will find the following Sample Information Library Specifications Data Volume Passing Filter and Aligned Base call Quality Score Distribution Coverage Summary Non N Reference Coverage Distribution SNV Indel Assessment Variant Statistics Structural Variants Summary nodey Aeuwuns Sample Information This section contains information associated with the sample from the included sample manifest Library Specifications This section describes details related to the library prep used in the sample Table 21 Library Specification Values Value Description Fragment Length Median Median fragment length of library sequence fragments calculated as for each pair of mapped reads For normal reads this value includes both reads along with the unsequenced insert between the reads Fragment Length SD The standard deviation of fragment lengths around the median Read Length Read lengths used in the build Read Type Will be par
19. strand for the array alleles relative to the reference A dash denotes a reverse compliment GC The GenCall score from the genotyping SNP call 0 15 cut off applied by default GT Genotype per VCF specification Whole Genome Sequencing Services User Guide 1 O BuidA 0u95 Analysis Deliverables Table gt FORMAT Fields ID GC GT Table 6 FILTER Fields ID GTEX NOCALL Description The GenCall score from the genotyping SNP call 0 15 cut off applied by default Genotype per VCF specification Description The exclude genotype filter The genotype was excluded in the mapping possibly because the probe failed to find a reference map failed to map uniquely or was an intensity only based probe Genotype value was not called on array Sample_Barcode GenotypingReport txt This file contains the genotyping report that is output from the GenomeStudio Genotyping Module Ilumina provides the genotyping report as a tab delimited text file and includes a header followed by at least the following columns Table 7 Genotyping Report Columns Column Allele1 Design Allele1 Forward Allele2 Design Allele2 Forward GC Score Sample Barcode SNP Name 11 Description The A allele call that is relative to the probe The A allele call that is relative to the submitted sequence The B allele call that is relative to the probe The B allele call that is relative to the submitted sequence The
20. 30 2012 09 05 2013 05 28 2013 06 16 2012 07 01 2013 06 07 2013 12 06 2009 Part 15040892 Rev D Technical Assistance For technical assistance contact Illumina Technical Support Table 30 Ilumina General Contact Information Website www illumina com Email techsupportGillumina com Table 31 Ilumina Customer Support Telephone Numbers Region Contact Number Region Contact Number North America 1 800 809 4566 Italy 800 874909 Australia 1 800 775 688 Netherlands 0800 0223859 Austria 0800 296575 New Zealand 0800 451 650 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 Whole Genome Sequencing Services User Guide 4 T 9UB SISSY EDIUYD9 A a E T TTT FGATAACAGTAACACACTTCTGTTAACCTT TTGTTGATCCACTGATTCAACGTACCGTATCAAT TGAGACTAAATAT TAACGTACCAI TAAGAGCTACCGTCTTCTGTTAACCTTAAGAT TACT TGATCCACTGAT TCAACGIACCGI CACTGAI CARGO TACONAGA TACT IGATGCACT GAT I CAAGG TACUGTAACGAACGTAT CAAT GAGACTARATATTAAGGTACCA TA GAGCTAGOGTGTTOI
21. 4 39 0 O Part 15040892 Rev D chr20 676575 AT 555 00 PASS SNVSB 50 0 SNVHPOL 3 GT GQ GQX DP DPF AD 1 1 114 114 39 0 0 39 chr20 676576 T 0 00 PASS END 676625 BLOCKAVG min30p3a GT GOX DP DPF 0 0 95 36 0 chr20 676626 T 0 00 PASS END 676650 BLOCKAVG min30p3a GT GQX DP DPF 0 0 117 40 0 chr20 676651 T 0 00 PASS END 676698 BLOCKAVG min30p3a GT GOX DP DPF 0 0 90 31 0 chr20 676699 T 0 00 PASS END 676728 BLOCKAVG min30p3a GT GOX DP DPF 0 0 69 24 0 chr20 676729 C 0 00 PASS GT GOX DP DPF 0 0 57 20 0 chr20 676784 C 0 00 PASS END 676803 BLOCKAVG min30p3a GT GOX DP DPF 0 0 51 18 0 chr20 676804 GA 62 00 PASS SNVSB 7 5 SNVHPOL 2 GT GQ GQX DP DPF AD 0 1 95 62 17 0 11 66 chr20 676805 0 00 PASS END 676818 BLOCKAVG min30p3a GT GOX DP DPF 0 0 48 17 0 chr20 676819 T 0 00 PASS END 676824 BLOCKAVG min30p3a GT GQX DP DPF 0 0 39 14 0 chr20 676825 A 0 00 PASS END 676836 BLOCKAVG min30p3a GT GQX DP DPF 0 0 30 11 0 chr20 676837 T 0 00 LowGQX END 676857 BLOCKAVG min30p3a GT GQX DP DPF 0 0 21 8 0 chr20 676858 G 0 00 PASS END 676873 BLOCKAVG min30p3a GT GQX DP DPF 0 0 30 11 0 D 676783 BLOCKAVG min30p3a E In addition to the nonvariant and variant regions in the example there is also 1 nonvariant region from 676837 676857 that is filtered out due to insufficient confidence that the region is homozygous reference Conventions
22. A e ee to i Ee A S git Trea TE k 7 PN x vera j Se caco ee ome Kae AP ponen 7 E 3 A Lord Sea Whole Genome Sequencing Services User Guide 1 Getting Started Whole Genome Sequencing Service The Whole Human Genome Sequencing Service Informatics Pipeline leverages a suite of proven algorithms to detect genomic variants comprehensively and accurately High quality sequence reads are aligned using the Isaac Aligner see Isaac Aligner on page 26 for details Variant calling is performed using the Isaac Variant Caller see Isaac Variant Caller on page 28 for details Two complementary approaches enable detection of large structural variations Read depth analysis by Isaac Copy Number Variant Caller See Isaac Copy Number Variant Caller on page 35 Discordant paired end analysis by Isaac Structural Variant Caller Manta See Isaac Structural Variant Caller on page 40 Identified variants are then annotated and compiled into a summary PDF This document provides an overview of the source and contents of the main files that Illumina creates using this informatics pipeline This document also provides information about key algorithms such as the Isaac Variant Caller Isaac SV Caller and Isaac CNV Caller The aim of this document is to help you understand the Whole Genome Sequencing data package that you receive from Ilumina The following versions of software packages are utilized in the Control Software CS v4 0 2 p
23. AACGTATCAAT TGAGACTAAATAT TAACGTACCAI TAAGAGCTACCG TGCAACGACGAAAAGAATGAT GATAACAGTAACACACI TOTGTTAACC T TAAGAT TACTTGATCCACTGAIT TCAACGTACCGTAACGAACGTATCAATTGAGACT AAATATIAACGTACCATIAAGAGCIACCOTCHOTOTT AACCTTAAGA TTACITGATOCACI GATTCAACI AA OASE SAIA N COAST TN CR AAA AKU b Alado CGTTAAGA R RN AAB H EAL CGTAACGAACG TATCAATTGAGCTICTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAAT TGAGACTAGCAACGACC GAAAAGAATGATAACAGTAACACACTICTGTTAACCTIAAGATTACTTGA TOGA CT GATT CAACGTACCGTAAAGATTACT TGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATATTAACGTACCAT TAAGAGCTAC Bat ea Ue og CA RPA LAE SR aT eA AS ATTER og AS oO A AE a BELLS OU pig Ua Fag CERES a aa TET Mg gol UN ng LL ES dae GTACCGT A E AEA GATTCAACGTACCGTAACGAACGTA ATE CTAAATAT TAACGTACCAT TAAGAGCTA EET GAR ie GUTO AA AOAC AGL TE Talc TT AAAAGAATGAT AACACACTTCTGTTAACCT TAAGAT TACT TGAI CCACTGAT TCAACGTACCGTAAAGAT TACT TGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAG AI TAACGTACCATTAAGAGCTAC ACORITAAGAGO ACO TGCAACAGTAACAGACITGTGI TAACOTTA GA TIACITGATOCACTGATTOMACGTACCGTAA GAAGGTAT CAATIGAGAGTABATATTAACGTACCAMAAGAGGTACCCTACAACCACGAAAR SANT GATA GATAACAG TAACACA EAS GI T DL AS RNA COPA CTAAATATTAACGTACCATTAAGAGCTACCGTCTICTGTTAACCTTAAGATTACTIGATCCACTGATTCAAC CTTGATCCACTGATTCAACGTIAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGCTICTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGT o AET CAAT TGAGACT SMS GTAOGAT TA GAGIU CTGTTAACCTTAAGA ACACIA TGAACG TACOGTAACGAA GT T CAATI GAGA CTAAATATTAACGTAC
24. ATTCAACGTACCGTAACGAACGTATCAATTGAGACTAACGACG AGACTAAATATTAACGTACCAT TAAGAGCTACAACCT TAAGAT TACT TGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATAT TAACGTACCATTAAGAGC TACCGT GCAACGACGAAAAGAAT GATAACAGTAACAC Se SK AACA L A di A CGTACCGTAACGAACGTATCAATTGAGACTAAATAT TAACGTACCATTAAGAGCTACCGTCTTCT PERLE SAE EEUU RS Og PA LOL TACCATTAAGAGC TACCGTGCAACTIAACCTIAAGAT TACTTGATCCACT GATT CAACGTACCGTAACGAACGTAT CAAT I GAGACTAAATATTAACGTACCATTAAGAGCTACCGTGCAACGACGAACTICTGTIAACCTIAAGATIACTTGA No TA PAPA AACA CR EE L GR E A era aera TAARE DARE AT SRO TGS GAAAAG GAAAAGAATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAAAGATTACTTGATO TGATAACAGTAACACACTTCTGTTAACCTTAAGAT TACTTGATCCACTGAT TCAACGTACCGTAACGAACGTATCAATTGAGACTA IACCAT TAAGAGCTACCGTGCAACAGTAACACACTTCTGT TAACCTTAAGAT TACTTGATCCACTGAT TCAACGTACCGTAACGA TGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACT GAT TCAACGTA SOV AAA ATE SGTACCGTAACGAACGTATCATTAAGATTACTT GATCCACT GATT CAACGTACCGTAACGAACGTAT CAATT GAGACTAAATAT TAF CITGATOCACTG I ICAACGI TAAGATACII GAI CGACTGATI CAACGTACCGITAACGAACGTAI CAATI GAGCI TCTGTTAA GAAAAGAATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAAAGATTACTTGATO E O erOMATGATTAGACCAC TCACAAGGTTTACCACAA KY TACASTAOGTACKACA NTCMGCGAMMGACAGGTTACCAT FOR RESEARCH USE ONLY ILLUMINA PROPRIETARY Part 15040892 Rev D June 2015 This document and its contents are proprietary to Ilumina Inc and its affiliates Illumina and are intende
25. BitArrays to store observed alignments one BitArray for each chromosome Each BitArray length is the same as its corresponding chromosome length As the BAM file is read in Isaac CNV Caller records the position of the left most base in each alignment within the chromosome appropriate BitArray After all alignments in the BAM file have been read the BitArrays have a 1 wherever an alignment was observed and a 0 everywhere else After reading in the BAM file a masked FASTA file is read in one chromosome at a time This FASTA file contains the genomic sequences that were used for alignment Each 35 mer within this FASTA file is marked as unique or nonunique with uppercase and lowercase letters If a 35 mer is unique then its first nucleotide is capitalized otherwise it is not capitalized For example in the sequence acgtttaATgacgatGaacgatcagctaagaatacgacaatatcagacaa The 35 mers marked as unique are as follows ATGACGATGAACGATCAGCTAAGAATACGACAATA TGACGATGAACGATCAGCTAAGAATACGACAATAT GAACGATCAGCTAAGAATACGACAATATCAGACAA Isaac CNV Caller stores the genomic locations of unique 35 mers in another collection of BitArrays analogous to BitArrays used to store alignment positions Unique positions and nonunigue positions are marked with 1 s and 0 s respectively This marking is used as Whole Genome Sequencing Services User Guide 3 5 J9 29 uenea Jaquiny Ado peges Analysis Overview Cleaning 36 a mask to guarantee tha
26. CAATTGAGACTAAGCTACCGTGCAACGACGAAAAGAA CAAARGRATGAJAACAGIAACACAC L GIA IAAGC LTAAGAT TACT TGAT CAG GAT CAACGTADCG TAAAGATIACT TGAICCAGT GAT ICAAGGTACCG TAA COMAG IATGAAT GAGAG TAAATAT TAAGGIACGAI TAAGAGCTAG AAG ARRON CTTCTGTTAACCTTAAGA RREO RAG AML TAC ACCGTAACGAACGTATCAATTGAGACTAAATATTAACGTACCA IIS EO A CGACGAAAAGAAT GATAACAGTAACA S KATKA TACCAT TAAGAGOC TACCGTGCAACAGTAACACACIT TI AAGATTACTTGATCCACTGATTCAACGTA OCGTAACGAACCI TAT AATTG A CTAAATA GTACCATTAAGAGCTACCGTGCAACGACGAAAAGAATGAT TGATAACAGTAACACACTTCTGTTAACCTTAAG ATACITGATO ACIGATTOAA GTA CCGTAACGAACGTATCAATTGAGACT NARTATIAACGTACCAIAAGAGCIACCOTCHCT GTTAACCTTAAGA FRAC I GATCCACTGATIGAAG l peg eI GUS AN a SAKT CH abe era OSKE GULA Nery ab O PTTL H tate NAE N CE AACGTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAAT TGAGCTTCTGTTAACCTTAAGAT TACTTGATCCACII aA dia in ola ATCAAT TGAGACTAGCAACGAC GAAAAGAATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGAT SGA CTGATTCAACGTACCGTAAAGATTACTTGATCCACTGAT TCAACGTACCGTAACGAACGTATCAATTGAGACTAAATATTAACGTACCAT TAAGAGCTAG cd e OSE UA EL GLa a A O A CI E rn v Sy Ns RC eee CAL ee WAG TER GCA A GATTORACOT ACCGTAACGAACGTA ARA CTAAATAT TAACGTACCAT TAAGAGCTACCGTGCAACGA Mate TO ppa Vesey CTTCTGTTAACCT GAAAAGAATGATAACAGTAACACACTTCTGTTAACCT TAAGAT TACTTGATCCACTGATTCAACGTA AAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAA TTAACGTACCATTAAGAGCTAC PAGCAT TARGAGO TAGUGT GUAACAGTAAGACACT CTO TAAGGT TAAGATIACT TGATOGAGTGAJ GAACGTAGUGTAACGAAGGTATCAATGAGAGAAATAT T TGATAACAGTAACA
27. CACTTCTGTT papel AAGATTACTTGATCCACTGATTCAACGTACCGT pecs CGTATCAATTGAGACTAAATATTAACGT A AHE GTTAACCTTAAGATTACTTGATCCACTGATTCAAC CTTGATCCACTGATTCAACGT TAAGAT TACTTGATCCACT GATT CAACGTACCGTAACGAACGTATCAAT TGAGCT TCT GT TAACCT TAAGAT TACTTGATCCACTGAT TCAACGTACCGT net eee LGR CU GA vey V ee ge CAAT TGAGACTAAATATTAACGTACCAT TAAGAGTCTGTTAACCTTAAGA TRAC TIGATCC AGT GATT CANCE TACOGTAACGAA G TATO AT TGAG CII AAATATTAACGTACCAT TAAGAGCTACCGTGCAACGAAAAGAAT GATAACAGT AS TOK TATRA SAT CCA NST UI Nay Sree SOA TRE AC A LN Ra DN H LCA EKO CTTGATCCACTGATTCAACGTTAAGAT TACTTGATCCACTGATT CAACGTACCGTAACGAACGTAT CAATTGAGCTTCTGTTAACCT TAAGAT TACT TGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGACTAGCAACGACI GAAAAGAATGAT TGTTAACCTTAAG TT TACCGT CGTACCI GAACGTA GAGACT CGTACI GA CACTGAT TCAACGTACCAAGATTACTTGATCCACTGAT TCAACGTACCGTAACGAACGTATCAATTGAGACTAAATAI TAACGTACCAI TAAGAGCTA o ACCGTAACG AAAAGAATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTAC IGATAACAGTAACAGAGT ICIGI TAACCTTAAGATTAGT TGATOGACT GATT GAACGTAGGGTAACGAAGGTAT CAAT IGAGACTAAATAT AAC TAGCAT TAAGAGC IAGUGTCT TOTO IAAGCT TANGA TAG IGATCCAGTGATTGAAG ATTGAGACTAAATATTAACGTTGTTAACCTTAA is AU AS Out GATTCAACGTACCGTAACGAACGTATCAATT GAGACTAAATAT TAACGTACCATTAAGAGCTTCTGTTAACCT TAAGATTA Ren AT e i TATCAAT TGAGACTAAATAT TAACGTACT TAACCTTAAGAT TACT TGATCCACTGATTCAACGTACCGTAACGAACGTCTTCTGT TAACCTTAAGATTACTTGATCCACTG
28. CATTAAGAGCTACCG TGCAACGAAAAGAATGATAACAG1 do da NARA VACA CAG TOA TOR CTTGATCCACTGATTCAACGITAAGA H RH LAH AL CCGTAACGAACGTATCAATTGAGCTTCTGTTAACCT TAAGAT TACT TGATCCACTGAI TCAACGTACCGTAACGAACGTATCAAT TGAGAC TAGCAACGACG GAAAAGAATGATAACAGTAACACACT TCTGTTAACCT TAAGAT TACTT GATCCACT GATT CAACGTACCGTAAAGAT TACT TGATCCACTGAT TCAACGTACCGTAACGAACGTAT CAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTAC GATAACAGTAACACACT TCTGTIAACCTIAAGAT TACTTGTTGATCCACTGAT TCAACGTACCGTAT CAAT TGAGACTAAATAT TAACGTACCAI TAAGAGCTACCGTCTTCTGTTAACCTIAAGAT TACT TGATCCACTGATTCAACGTACCGI CACTGAT TCAACGI TEE A A r TOT CGTATCAATTGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGTCTTCTGTTAACCT TAAGAT TACTTGATCCACTGATTCAACGTACCGTAACG GAAAAGAATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAAT TGAGAC TAAATAT TAACGTACCATTAAGAGCTAC LNR Ra ATO TEA SA TO fo oO A ayy TETT RACIAN TAART NOS VH AA SAEC TE O TAR OTA ORA NS CO L y T TGAGACTAAATAT TAACGT TGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAATATTAACGTACCATTAAGAGCTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGT TATCAATTGAGACTAAATAT TAACGTACT TAACCTTAAGAI TACTTGATCCACTGATTCAACGTACCGTAACGAACGTCTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAAT TGAGACTAACGACG STA eM oe TA UN E ENS NU ov L OS RU UNESA Gea TENTA NA UM ARE ANA AC ORAVU V VAN SP L T GATAACAGTAACACACT TCTGTTAACCI TAAGATTACI TGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATAT TAACGTACCATTAAGAGCTACCGTCTTCTGTTAACCTTAA RR
29. NT EXCEED THE AMOUNT PAID TO ILLUMINA FOR THIS PRODUCT 7 Limitations on Illumina Provided Warranties TO THE EXTENT PERMITTED BY LAW AND SUBJECT TO THE EXPRESS PRODUCT WARRANTY MADE HEREIN ILLUMINA MAKES NO AND EXPRESSLY DISCLAIMS ALL WARRANTIES EXPRESS IMPLIED OR STATUTORY WITH RESPECT TO THIS PRODUCT INCLUDING WITHOUT LIMITATION ANY IMPLIED WARRANTY OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE NONINFRINGEMENT OR ARISING FROM COURSE OF PERFORMANCE DEALING USAGE OR TRADE WITHOUT LIMITING THE GENERALITY OF THE FOREGOING ILLUMINA MAKES NO CLAIM REPRESENTATION OR WARRANTY OF ANY KIND AS TO THE UTILITY OF THIS PRODUCT FOR PURCHASER S INTENDED USES 8 Product Warranty All warranties are personal to the Purchaser and may not be transferred or assigned to a third party including an affiliate of Purchaser All warranties are facility specific and do not transfer if the Product is moved to another facility of Purchaser unless Ilumina conducts such move a Warranty for Consumables Illumina warrants that Consumables other than custom Consumables will conform to their Specifications until the later of i 3 months from the date of shipment from Illumina and ii any expiration date or the end of the shelf life pre printed on such Consumable by Ilumina but in no event later than 12 months from the date of shipment With respect to custom Consumables i e Consumables made to specifications or designs made by Purchaser or provided to Il
30. Part 15040892 Rev D Table 18 INFO Fields ID AA CF1000G BLOCKAVG_ min30p3a CIGAR CLINVAR SON CSOR COSMIC END EVS GMAF IDREP phastCons REFREP RU SNVHPOL SNVSB Description The inferred allele ancestral to the chimpanzee human lineage The allele frequency from all populations of 1000 genomes data Nonvariant site block All sites in a block are constrained to be nonvariant 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 The CIGAR alignment for each alternate indel allele Clinical significance Transcript consequence as predicted by VEP version 72 using transcripts from Ensmbl Annotated as HGNC TranscriptID Consequence Regulatory consequence type as predicted by VEP version 72 using features from Ensmbl Annotated as RegulatoryID Consequence The numeric identifier for the variant in the Catalogue of Somatic Mutations in Cancer COSMIC database The end position of the region described in this record Allele frequency sample count and coverage taken from the Exome Variant Server EVS Annotated as AlleleFreqEVS EVSCoverage EVSSamples Global minor allele frequency GMAF technically the frequency of the second most frequent allele Annotated as GlobalMinorAllele AlleleFreqGlobalMinor Number of times
31. RU is repeated in an indel allele Denotes if the variant is an identical or similar sequence that occurs between species and maintained between species throughout evolution Number of times RU is repeated in the reference The smallest repeating sequence unit extended or contracted in the indel allele relative to the reference RUs are not reported if longer than 20 bases SNV contextual homopolymer length SNV site strand bias Whole Genome Sequencing Services User Guide 1 6 SUOIJENEN Analysis Deliverables 17 Table 19 FORMAT Fields ID AD DE DPF DPI GQ GQX GT Description Allelic depths for the ref and alt alleles in the order listed For indels this value includes only reads that confidently support each allele Specifically includes reads for which the posterior probability is 0 999 or higher that the read contains an indicated allele versus all other intersecting indel alleles Filtered base call depth used for site genotyping Base calls filtered from input before site genotyping Read depth associated with indel taken from the site preceding the indel Genotype quality Minimum Phred genotype quality Annotated as Genotype quality assuming variant position Genotype quality assuming nonvariant position Genotype Table 20 FILTER Fields ID Description HighDepth The locus depth is greater than 3x the mean chromosome depth HighDPFRatio The fraction of base calls filtered out at a
32. 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 Whole Genome Sequencing Services User Guide 2 9 191 0 JURUBA DLLs Analysis Overview Genome VCF gVCF Human genome sequencing applications require sequencing information for both variant and nonvariant positions yet there is no common exchange format for such data gVCF addresses this issue gVCF is a set of conventions applied to the standard variant call format VCF These conventions allow representation of genotype annotation and additional information across all sites in the genome in a reasonably compact format Typical human whole genome sequencing results expressed in gVCF with annotation are less than 1 7 GB or about 1 50 the size of the BAM file used for variant calling gVCF is also equally appropriate for representing and compressing targeted sequencing results Compression is achieved by joining contiguous nonvariant 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 such as depth and genotype quality reflect the minimum of any site in the block The gVCF file is also a valid VCF v4 1 file and can be indexed and used w
33. d 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 OUT OF THE IMPROPER USE OF THE PRODUCT S DESCRIBED HEREIN INCLUDING PARTS THEREOF OR SOFTWARE 2015 Illumina Inc All rights reserved Ilumina 24sure BaseSpace Bead Array BlueFish BlueFuse BlueGnome cBot CSPro CytoChip DesignStudio Epicentre GAIIx Genetic Energy Genome Analyzer GenomeStudio GoldenGate HiScan HiSeq HiSeq X Infinium iScan iSelect MiSeq MiSeqDx NeoPrep Nextera Nex
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35. e Sequencing Services User Guide 6 2 n19N 113 JOp 04 1NS9Y Analysis Deliverables Assembly The assembly folder contains the sequence data used to assemble the sample genome Sample Barcode bam The included archival BAM file contains all pass filter reads input into the analysis pipeline for a sample and includes aligned duplicate and unaligned reads To reduce the data storage footprint while not compromising accuracy Ilumina has reduced quality score resolution in BAM files In practice this means that the more commonly used 40 possible Q scores have been reduced to 8 bins This transformation is performed on instrument and is calibrated for each individual quality table For details about how Illumina has reduced storage requirements while maintaining compatibility and accuracy see the Reducing Whole Genome Data Storage Footprint white paper on the Illumina product literature page Sample_Barcode bam bai This is the SAMtools BAM index for the BAM file This file can be used with SAMtools and other tools utilizing the SAMtools specification for fast retrieval of targeted regions in the associated BAM file BAM File Details The included BAM file adheres to the SAM format specification wherever possible The following sections cover BAM file details that are not evident in the specification Singleton Shadow Pairs Read Groups RG Read name RNAME Bitwise Flag Notes FLAG Extended Tags Optional Fields MAPQ S
36. e across the genome defined as bases used in variant calling over autosomal regions total non N reference length of autosomal regions Non N Reference Coverage Distribution This histogram of coverage depth uses the same definition of coverage as the Coverage Summary SNV and Indel Assessment These tables provide the total number of SNVs and Indels overlapping known variants and genes exons and coding regions All counts only use PASS filter variants where 19 applicable Table 24 SNV and Indel Assessment Table Values Value Array Agreement in Coding in dbSNP in Exons in Genes Het Hom Description The percentage of concordant SNVs between the genotyping and sequencing SNVs Note Only PASS filter SNVs are compared Percent of PASS filter variants overlapping a coding position for any annotated transcript Percent of PASS filter variants that overlap a dbSNP identifier in annotation Percent of PASS filter variants overlapping a coding 5 UTR or 3 UTR position for any annotated transcript Percent of PASS filter variants overlapping a coding 5 UTR 3 UTR or intron position for any annotated transcript The ratio of heterozygous to homozygous PASS filter variants reported Part 15040892 Rev D Value Description Ti Tv The transition tranversion ratio for reported variants relative to the reference base or bases Total Total number of PASS filter SNVs reported
37. e effect is to flatten the midpoints of the bars in the example box and whisker plot Some values for GC content have few bins so the estimate of its median is not robust Therefore bins are discarded when the number of bins having the same GC content is fewer than 100 For some sample preparation schemes GC content correction has a dramatic effect The following figure illustrates the effect of GC content correction for a low depth sequencing experiment using the Nextera library preparation method The figure on the left shows bins counts as a function of chromosome position before normalization The figure on the right shows the result after GC content correction Analysis Overview NA12878_NXV2 Raw NA12878_NXV2 Normalized For whole genome sequencing experiments the typically median absolute deviations MADs are 10 3 which is close to the expected value of 10 The expected value is predicted using the Poisson model for an average count of 100 and indicates that little bias remains following GC content correction It is important to note that the normalization signal does not dampen signal from CNVs as shown in the following 2 figures The figure on the left shows a chromosome known to harbor a single copy gain The figure on the right shows chromosome known to harbor a double copy gain NA102 6 chr Mextera W2 Cometa De Lange Syndrome GM12508 chr7 Nextera V2 ay Z so E joe 5 100 co m m a postion mb postion mb
38. e terms and conditions iii the use of this Product in combination with any other products materials or services not supplied by Ilumina iv the use of this Product to perform any assay or other process not supplied by Illumina or v Ilumina s compliance with specifications or instructions for this Product furnished by or on behalf of Purchaser each of i v is referred to as an Excluded Claim Indemnification by Purchaser Purchaser shall defend indemnify and hold harmless Illumina its affiliates their non affiliate collaborators and development partners that contributed to the development of this Product and their respective officers directors representatives and employees against any claims liabilities damages fines penalties causes of action and losses of any and every kind including without limitation personal injury or death claims and infringement of a third party s intellectual property rights resulting from relating to or arising out of i Purchaser s breach of any of these terms and conditions ii Purchaser s use of this Product outside of the scope of research use purposes iii any use of this Product not in accordance with this Product s Specifications or Documentation or iv any Excluded Claim Conditions to Indemnification Obligations The parties indemnification obligations are conditioned upon the party seeking indemnification i promptly notifying the other party in writing of such claim or action
39. ed end for the standard Whole Genome Sequencing workflow Data Volume This table in the reports the volume of data input into the assembly process and in the associated BAM file Table 22 Data Volume Table Values Value Description Passing Filter Yield is the number of gigabases of data PASS filter data only input into the build Bases Q30 where Q30 is the percent of the sequence data that has a Q score of Q30 or greater Note Q score binning does not affect this measure Passing Filter and Same as passing filter but reported only for the subset of data that Aligned aligns to the genome Whole Genome Sequencing Services User Guide 1 8 Analysis Deliverables Passing Filter and Aligned Base call Quality Score Distribution This table details the quality score distribution for the aligned reads for a sample The spiky appearance of the graph is due to the effect of quality score binning Coverage Summary The coverage summary reports the distribution of depth of coverage across the genome Coverage is calculated from bases not flagged as duplicates and for which both read pairs map unambiguously Table 23 Coverage Summary Values Value gt 5 10 20x coverage Callable Average Coverage Description Number of non N reference autosomal positions that have greater or equal to 5 10 20 fold coverage The percent of autosomal non N reference genome in gVCF file with a PASS filter status Mean coverag
40. ements entered into and all final judgments and costs including reasonable attorneys fees awarded against Purchaser in connection with such infringement claim If this Product or any part thereof becomes or in Illumina s opinion may become the subject of an infringement claim Ilumina shall have the right at its option to A procure for Purchaser the right to continue using this Product B modify or replace this Product with a substantially equivalent non infringing substitute or C require the return of this Product and terminate the rights license and any other permissions provided to Purchaser with respect this Product and refund to Purchaser the depreciated value as shown in Purchaser s official records of the returned Product at the time of such return provided that no refund will be given for used up or expired Consumables This Section states the entire liability of Ilumina for any infringement of third party intellectual property rights Exclusions to Illumina Indemnification Obligations Illumina has no obligation to defend indemnify or hold harmless Purchaser for any Illumina Infringement Claim to the extent such infringement arises from i the use of this Product in any manner or for any purpose outside the scope of research use purposes ii the use of this Product in any manner not in accordance with its Specifications its Documentation the rights expressly granted to Purchaser hereunder or any breach by Purchaser of thes
41. er Variant Caller respectively Revised documentation to reflect changes in version 3 of the Ilumina FastTrack WGS pipeline Added Circos plot legend plus minor modifications Initial release Whole Genome Sequencing Services User Guide Part 15040892 Rev D Table of Contents Revision History c en V Table of Contents vii Chapter 1 Getting Started 1 Whole Genome Sequencing Service o oocccccccccccccccccccccccncccccncccnnncccno 2 Data Delivery ooo ooo 3 Chapter 2 Analysis Deliverables nn 4 Analysis Folder Structure Overview o occcccccccccccccccccccccccnncccccccccnnocos 5 Result Folder Structure oi 6 Assembly rollo zs i n os 7 ELA AAA A alee and cs AA 10 E A A A doa AR RR E eee Spa 12 Summary Report eee 18 Data Integrity llc nn n eee cnn 22 Chapter 3 Analysis Overview eee 23 asees T 24 Genome Specific Details 25 Isaac Aligner 2 0 0 ooo 26 Isaac Variant Caller 28 Genome VCF gVCF 2 2220 30 Isaac Copy Number Variant Caller 35 Isaac Structural Variant Caller 40 Appendix A Appendix go ori 43 BAM File FAQ HRE rai 44 Illumina FastTrack Services Annotation Pipeline 46 Technical Assistance 47 Whole Genome Sequencing Services User Guide VI Part 15040892 Rev D Getting Started Whole Genome Sequencing Service LJISIdeuyo Data Delivery AN 3 a ker a Ne p y n z E ces cana SS 4 as E ER AAA Es 0 S E S AAAA
42. eriod on the original Hardware warranty whichever is shorter If only a component is being repaired or replaced the warranty period for such component is 90 days from the date of shipment or the remaining period on the original Hardware warranty whichever ends later The preceding states Purchaser s sole remedy and Illumina s sole obligations under the warranty provided hereunder Third Party Goods and Warranty Illumina has no warranty obligations with respect to any goods originating from a third party and supplied to Purchaser hereunder Third party goods are those that are labeled or branded with a third party s name The warranty for third party goods if any is provided by the original manufacturer Upon written request Ilumina will attempt to pass through any such warranty to Purchaser Indemnification a Infringement Indemnification by Illumina Subject to these terms and conditions including without limitation the Exclusions to Ilumina s Indemnification Obligations Section 9 b below the Conditions to Indemnification Obligations Section 9 d below Illumina shall i defend indemnify and hold harmless Purchaser against any third party claim or action alleging that this Product when used for research use purposes in accordance with these terms and conditions and in accordance with this Product s Documentation and Specifications infringes the valid and enforceable intellectual property rights of a third party and ii pay all settl
43. escription BC Number of bins in the region CN Copy number genotype for imprecise events GT Genotype RC Mean counts per bin in the region Table 11 FILTER Fields ID Description g10 Ouality below 10 L10kb For a small variant lt 1000 base the fraction of reads with MAPQ 0 around either break end that exceeds 0 4 Sample Barcode SNPs vcf gz and Sample Barcodel Indels vcf gz Sample Barcode SNPs vcf gz and Sample Barcode Indels vcf gz The SNV and inde files list the single nucleotide polymorphisms and indels respectively that were called by the Isaac Variant Caller Small indels are limited to 50 bp The VCF file contains the following fields Whole Genome Seguencing Services User Guide 1 P SUOIJENE AN Analysis Deliverables 13 Table 12 INFO Fields ID CIGAR END RU IDREP REFREP SNVHPOL SNVSB Description The CIGAR alignment for each alternate indel allele The end position of the region described in this record The smallest repeating sequence unit extended or contracted in the indel allele relative to the reference RUs are not reported if longer than 20 bases Number of times RU is repeated in an indel allele Number of times RU is repeated in the reference SNV contextual homopolymer length SNV site strand bias Table 13 FORMAT Fields ID AD DP DPF DPI GQ GOX GT OPL Description Allelic depths for the ref and alt alleles in the order listed For indels t
44. f biomarkers or sequences are examples of Application Specific IP Consumable s means Illumina branded reagents and consumable items that are intended by Illumina for use with and are to be consumed through the use of Hardware Documentation means Illumina s user manual for this Product including without limitation package inserts and any other documentation that accompany this Product or that are referenced by the Product or in the packaging for the Product in effect on the date of shipment from Illumina Documentation includes this document Hardware means Ilumina branded instruments accessories or peripherals Ilumina means Illumina Inc or an Illumina affiliate as applicable Product means the product that this document accompanies e g Hardware Consumables or Software Purchaser is the person or entity that rightfully and legally acquires this Product from Illumina or an Ilumina authorized dealer Software means Illumina branded software e g Hardware operating software data analysis software All Software is licensed and not sold and may be subject to additional terms found in the Software s end user license agreement Specifications means Illumina s written specifications for this Product in effect on the date that the Product ships from Ilumina 2 Research Use Only Rights Subject to these terms and conditions and unless otherwise agreed upon in writing by an officer of Ilumina Purchaser is granted only a non exclusiv
45. fields in the BAM file Whole Genome Sequencing Services User Guide Analysis Deliverables Table 3 BAM File Fields Field AS BC NM GT RG SM Description Pair alignment score Barcode string Edit distance mismatches and gaps including the soft clipped parts of the read Original CIGAR for the realigned reads Isaac read groups correspond to unique flow cell lane barcodes Single read alignment score Mapping Quality MAP G For pairs that match the dominant template orientation the MAPO value in the AS field is capped For reads that are not members of a pair matching the dominant template orientation the MAPO value in the SM field is capped at 60 The MAPO could be downgraded to O or set to be unknown 255 for alignments that do not have enough evidence to be correctly scored Part 15040892 Rev D Genotyping If available variants called using the Infinium platform are compared to sequencing calls to confirm identity and make sure that data are of high quality This folder contains the results of the genotyping SNP calls and the necessary files needed to regenerate them To download the end user documentation for the GenomeStudio Genotyping Module go to support illumina com documents Mylllumina d2c2c169 36c7 4613 89d6 bf34588a7624 GenomeStudio GT Module v1 0 UG 11319113 RevA pdf Sample Barcode idats This folder contains the GRN idat and RED idat intensity files and the sample sheet for a genotyping sam
46. he following issues when joining adjacent nonvariant sites into block records The criteria that allow adjacent sites to be joined into a single block record The method to summarize the distribution of SAMPLE or INFO values from each site in the block record At any gVCF compression level a set of sites can be joined into a block if Each site is nonvariant with the same genotype call Expected nonvariant genotype calls are 0 0 0 453 Each site has the same coverage state where coverage state refers to whether at least 1 read maps to the site For example sites with 0 coverage cannot be joined into the same block with covered sites Each site has the same set of FILTER tags Sites have less than a threshold fraction of nonreference allele observations compared to all observed alleles based on AD and DP field information This threshold is used to keep sites with high ratios of nonreference alleles from being compressed into nonvariant blocks In the Isaac Variant Caller gVCF output the maximum nonreference fraction is 0 2 Block Sample Values Any field provided for a block of sites such as read depth using the DP key shows the minimum observed value among all sites encompassed by the block Nonvariant Block Implementations Files conforming to the gVCF conventions delineated in this document can use different criteria for creation of block records depending on the desired trade off between compression and nonvariant site de
47. his value includes only reads that confidently support each allele Specifically includes reads for which the posterior probability is 0 999 or higher that the read contains an indicated allele versus all other intersecting indel alleles Filtered base call depth used for site genotyping Base calls filtered from input before site genotyping Read depth associated with indel taken from the site preceding the indel Genotype quality Minimum Phred genotype quality Annotated as Genotype quality assuming variant position Genotype quality assuming nonvariant position Genotype Original Phred scaled genotype likelihood PL value before ploidy correction Only applies to sites with HAPLOID_CONFLICT FILTER applied Table 14 FILTER Fields ID GENDER_ CONFLICT HAPLOID_ CONFLICT HighDepth Description Genotype is inconsistent with sample gender Locus has heterozygous genotype in a haploid region The locus depth is greater than 3x the mean chromosome depth Part 15040892 Rev D ID HighDPFRatio HighSNVSB IndelConflict LowGOX SiteConflict Description The fraction of base calls filtered out at a site is greater than 0 3 SNV strand bias value SNVSB exceeds 10 The locus is in a region with conflicting indel calls Locus GQX is less than 30 or not present The site genotype conflicts with the proximal indel call This is typically a heterozygous SNV call made inside a heterozygous de
48. ighbors then the bin is deemed an outlier and removed Physical Size Outlier Removal Bins likely do not have the same physical genomic size The average for whole genome sequencing runs might be approximately 1 kb If the bins cover repetitive regions of the genome some bins sizes might be several megabases in size Example regions might include centromeres and telomeres The counts in these regions tend to be unreliable so bins with extreme physical size are removed Specifically the 98th percentile of observed physical sizes is calculated and bins with sizes larger than this threshold are removed GC Content Correction The main variability in bins counts is GC content An example of the bias is represented in the following figure Figure 2 GC Bias Example GC Bias Following CanvasBin n 2 c 3 o o D E 10 a O 4 1 TTTTTTTTITITTTTITITITTIT TTTTTTTTTTITITTITTITITITITITITITITITITITITITITTITITITITITITITTIT O 4 8 18 19 25 31 37 43 49 65 61 67 73 79 65 GC Content The following correction is performed 1 Bins are first aggregated according to GC content which is rounded to the nearest integer 2 Second each bin count is divided by the median count of bins having the same GC content Whole Genome Sequencing Services User Guide 3 J9 29 JUBUBA Jaquiny Ado oees 3 Finally this value is multiplied by the desired average count per bin 100 by default and rounded to the nearest integer Th
49. iling quality checks Indel calling Identifies a set of possible indel candidates and realigns all reads overlapping the candidates using a multiple sequence aligner SNV 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 Indel Candidates Input reads are filtered by removing any of the following Reads that failed base calling 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 summarization by the SNV caller SNV Calling The variant caller runs a series of filters on the set of filtered and realigned reads for SNV 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 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 2 mismatches to the reference sequence within 20 ba
50. illumina Whole Genome Sequencing services User Guide ESA VT TR O Ula ea OL L eG AOU IAL gw TCA A EO VY NE POTU A MMs GEC Ap Olly call SS GTACCATTAAGAGCTAC TGATAACAGTAACACACTTCTGTTAACCTTAAGAT TACTTGT TGATCCACTGATTCAACGTACCGTATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGTCTTCTGTTAACCTIAAGATTACTTGATCCACTGATTCAACGTACCG IGAGT GAT TCAACGTACCAAGAT TACT IGATCCACT GAT IGAAOGTAGGGT R ae a CR AO CLA TA ATON TOANDE eee eee GAAAAGAATGATAACAGTAACACACTTCTGTTAACCTT UVAS O TYM TACCG TO OO NTE CAACGTACCGTAACGAACGTATCAATTGAGACTAAATATTAACGTACCAT TAAGAGCTACI TGATAACAGTAACACACTTCTGTTAACCTTAAGATT ACTTO GATCCACTGATTCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATATTAACGTACCATTAAGAGCTACCGTCTTCTGTTAACCT TAM ATH ACTTGATCCACTGATTCAAC ITIGAGACTAAATAT AGG LG TAACCT TAAGAT TACT GATCCACTGAI IGAAGG TACCGTAACGAACG IAT CAAT IGAGACTAAATATIAACGTACOAT IAAGAGC TT T Ru LU TOT VN LY HAATI E TGATTCAACGT ee AACGAACGTCTICTGTTAACCTTAAGAT T R AEN Lea PAA GTA ANA ARG TCAA IRAGA A TG CGTACCAI TAAGAGCTACAACCTT TTGATO A TGATTCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATATTAACGTACCATTAAGAGCTACCGTGCAACGACGAAAAGAA CAGTAACAC IGATAACAGTAAGAGAGT TGIG MARCO TAGAT TACT IGATGUAG GAT TOMOS IACGG TAACGAAGG Tal CAAT GAGACAAATAT TAAGG IACOR TRAGAGCIACOGIGII CT TARO TAAGATTACI TGATOCACI CAT TORAC Aa ANNE ET UL GATTACTTGATCCACT ALC PACA N feelin eV TS AAAT E NO TTAAGAGCTACCGTGCAACGACGAACT TCTGT TAACCTTAAGATTACI TI a GCTACCGTGCAACGAAAATAA AAGATTACT TGAI CCACTGAI TCAACG AACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTAT
51. ingleton Shadow Pairs Singleton shadow pairs refer to pairs for which the aligner was unable to determine the alignment of 1 of the ends The determined end is the singleton and the undetermined end is the shadow Shadows are assigned the position of the end that does align To maintain SAMtools format compatibility the shadows are stored in the BAM file immediately after their respective singletons with CIGAR empty and corresponding flag 4 set Shadows can be retrieved using the following SAMtools command samtools view f 4 input bam gt output sam Read Groups RG Where possible unique flow cell lane index mappings split up the read groups in the BAM The following is an example from a BAM header RG ID 0 PL ILLUMINA SM NA12878 PU COLOAACXX 1 none RG ID 1 PL ILLUMINA SM NA12878 PU COL54ACXX 7 none RG ID 2 PL ILLUMINA SM NA12878 PU COL54ACXX 8 none Part 15040892 Rev D In the example the read group 0 is derived from the flow cell barcode ID COLOAACXX lane 1 without a specified index for sample NA12891 In this example read groups 1 and 2 are from a different flow cell COL54ACXX lanes 7 and 8 Read name RNAME The read name consists of the following pattern detailing the flow cell lane and tile on which the sample was run flowcell id lane number cluster id alt mem Table 1 RNAME Variables ID Description cluster id within the tile flowcell id Flow cell barcode cluster id alt tile n
52. ipeline Software Version Purpose Isaac Aligner 6 15 01 Align reads to the human hg19 reference Isaac Variant Caller 2 1 4 Germline SNV and indel caller Isaac Copy Number Variant 1 1 0 Germline copy number variant caller Caller Isaac Structural Variant Caller 0 23 1 Germline and somatic structural variant caller Part 15040892 Rev D Data Delivery Ilumina FTS currently provides data delivery through the following choices Ilumina Hard Drive Data Delivery Illumina FastTrack Services ships data on 1 or more hard drives The hard drives are formatted with the NTES file system and can optionally be encrypted The data on the hard drive are organized in a folder structure with 1 top level folder per sample or analysis Ilumina Cloud Data Delivery Illumina FastTrackServices uploads data to a cloud container Ilumina currently supports uploads to the Amazon S3 service Upload data are organized per upload batch by date under an Ilumina FTS prefix For example a sample in a batch uploaded on February 1 2014 would be found with the prefix Ilumina FTS 20140201 SAMPLE BARCODE in the container Contact your FastTrack Services project manager to enable cloud delivery Whole Genome Sequencing Services User Guide 3 lam aq e1eQ Analysis Deliverables Analysis Folder Structure Overview ooo 5 Result Folder Structure 6 A PA hn A I SG O Ae ate oie ES K i ree o haa 7 GenotVPIAG soc et a s SN a eet tocata 10 Vanations casos
53. ith existing VCF tools such as tabix and IGV This feature makes the file convenient both for direct interpretation and as a starting point for further analysis gvcftools Illumina has created a full set of utilities aimed at creating and analyzing Genome VCF files For up to date information and downloads visit the gvcftools website at sites google com site gvcftools home Examples The following is a segment of a VCF file following the gVCF conventions for representation of nonvariant sites and more specifically using gvcftools block compression and filtration levels In the following gVCF example nonvariant regions are shown in normal text and variants are shown in bold d NOTE i The variant lines can be extracted from a gVCF file to produce a conventional variant VCF file chr20 676337 T 0 00 PASS END 676401 BLOCKAVG min30p3a GT GOX DP DPF 0 0 143 51 0 chr20 676402 A 0 00 PASS END 676441 BLOCKAVG min30p3a GT GOX DP DPF 0 0 169 57 0 chr20 676442 T G 287 00 PASS SNVSB 30 5 SNVHPOL 3 GT GQ GQX DP DPF AD 0 1 316 287 66 1 33 33 chr20 676443 T 0 00 PASS END 676468 BLOCKAVG min30p3a GT GOX DP DPF 0 0 202 68 1 E chr20 676469 G 0 00 PASS GT GOX DP DPF 0 0 199 67 5 chr20 676470 A 0 00 PASS END 676528 BLOCKAVG min30p3a GT GOX DP DPF 0 0 157 53 0 chr20 676529 T 0 00 PASS END 676566 BLOCKAVG min30p3a GT GOX DP DPF 0 0 120 41 0 chr20 676567 C 0 00 PASS END 676574 BLOCKAVG min30p3a GT GOX DP DPF 0 0 11
54. ivided into 2 sections 1 describes filtering based on genotype quality 2 describes all other filters S NOTE These filters are default values used in the current Isaac Variant Caller implementation However no set of filters or cutoff values are required for a file to conform to gVCF conventions The genotype guality is the primary filter for all sites in the genome In particular traditional discovery based site guality values that convey confidence that the site is anything besides the homozygous reference genotype such as SNV guality are not used Instead a site or locus is filtered based on the confidence in the reported genotype for the current sample The genotype quality used in gVCF is a Phred scaled probability that the given genotype is correct It is indicated with the FORMAT field tag GOX Any locus where the genotype quality is below the cutoff threshold is filtered with the tag LowGQX In addition to filtering on genotype quality some other filters are also applied The gVCF output from Isaac Variant Caller includes several heuristic filters applied to the site and indel records The filters are as follows Table 28 VCF Site and Indel Record Filters VCF Filter ID Type Description HAPLOID_ site indel Locus has heterozygous genotype in a haploid region CONFLICT HighDepth site indel The locus depth is greater than 3x the mean chromosome depth HighDPFRatio site The fraction of base calls filtered out at a site i
55. letion Sample_Barcode SV vcf gz The SV file contains structural variants from 50 bp 10 kb called within the sample by the Isaac SV Caller The VCF file contains the following fields Table 15 ALT Fields ID BND COMPLEX DEL DUP TANDEM INS INV Table 16 INFO Fields ID BND_DEPTH BND_PAIR_COUNT CIEND CIGAR CIPOS DOWNSTREAM_ PAIR_COUNT END HOMLEN Description Translocation break end Unknown Candidate Type Deletion Tandem Duplication Insertion Inversion Description Read depth at local translocation break end Confidently mapped reads supporting this variant at this break end it is possible that mapping is not confident at remote break end Confidence interval around END CIGAR alignment for each alternate indel allele Confidence interval around POS Confidently mapped reads supporting this variant at this downstream break end it is possible that mapping is not confident at upstream break end End position of the variant described in this record Length of base pair identical micro homology at event breakpoints Whole Genome Sequencing Services User Guide 1 4 SUOI BLCA Analysis Deliverables ID HOMSEQ IMPRECISE MATE BND DEPTH MATEID PAIR COUNT SVINSLEN SVINSSEQ SVLEN SVTYPE UPSTREAM UPSTREAM PAIR COUNT Table 17 FORMAT Fields Description Sequence of base pair identical micro homology at event breakpoints Imprecise structural variation Read depth a
56. ls 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 from the extremities of the read as seeds Isaac Aligner performs another search using different seeds for only those reads that were not mapped unambiguously with the first pass seeds 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 score 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 MAPQ is the alignment score truncated to 60 for scores above 60 and c
57. lumina by or on behalf of Purchaser Illumina only warrants that the custom Consumables will be made and tested in accordance with Illumina s standard manufacturing and quality control processes Ilumina makes no warranty that custom Consumables will work as intended by Purchaser or for Purchaser s intended uses b Warranty for Hardware Ilumina warrants that Hardware other than Upgraded Components will conform to its Specifications for a period of 12 months after its shipment date from Illumina unless the Hardware includes Illumina provided installation in which case the warranty period begins on the date of installation or 30 days after the date it was delivered whichever occurs first Base Hardware Warranty Upgraded Components means Illumina provided components modifications or enhancements to Hardware that was previously acquired by Purchaser Illumina warrants that Upgraded Components will conform to their Specifications for a period of 90 days from the date the Upgraded Components are installed Upgraded Components do not extend the warranty for the Hardware unless the upgrade was conducted by Illumina at Illumina s facilities in which case the upgraded Hardware shipped to Purchaser comes with a Base Hardware Warranty c Exclusions from Warranty Coverage The foregoing warranties do not apply to the extent a non conformance is due to i abuse misuse neglect negligence accident improper storage or use contrary to the Documentation or
58. mplied by the classification For instance a variant marked as a deletion by Isaac SV Caller indicates an intrachromosomal translocation with a deletion like break end pattern However there is no test of depth b allele frequency or intersecting adjacencies to infer the SV type directly Whole Genome Sequencing Services User Guide 41 J9 29 JUBULA EJNI2NJIS 9ees AZ Part 15040892 Rev D Appenalx BAM File FAQ anaa Ilumina FastTrack Services Annotation Pipeline Whole Genome Sequencing Services User Guide 43 Appendix BAM File FAQ A large volume of data represents the sequence and corresponding alignments which are provided in BAM format There are a few methods to convert BAM into different formats such as FASTO files Picard Tools FASTQ Extraction Many pipelines start from FASTO files To convert BAM files to FASTO files using Picard tools refer to the following example Convert bam into readl fastq and read2 fastg Sjava jar picard tools 1 110 SamToFastq jar INPUT Example bam FASTO Example Rl fastg SECOND END FASTO Example R2 fastq VALIDATION STRINGENCY SILENT BAM Size 79 G Wall Clock Time 3 hrs 54 min Optional arguments RE REVERSE true Reverts the sequence to the native orientation Otherwise all aligned sequence is forward orientation MAX RECORDS IN RAM 5000000 Decides the number of reads held memory and controls total memory usage
59. n any region where overlapping deletion evidence cannot be resolved into 2 haplotypes all indel and set records in the region are marked with the IndelConflict filter Table 27 Indel Conflict Filters ID Type Description IndelConflict site indel Locus is in region with conflicting indel calls SiteConflict site Site genotype conflicts with proximal indel call This conflict is typically a heterozygous genotype found inside a heterozygous deletion Representation of Non Variant Segments 32 This section includes the following subsections Block representation using END key Joining nonvariant sites into a single block record Block sample values Nonvariant block implementations Block Representation Using END Key Continuous nonvariant 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 Following is a simplified example of a nonreference block record INFO lt ID END Number 1 Type Integer Description End position of the variant described in this record gt CHROM POS ID REF ALT QUAL FILTER INFO FORMAT NA19238 Part 15040892 Rev D chr1 51845 A PASS END 51862 The example record spans positions 51845 51862 Joining Non Variant Sites Into a Single Block Record Address t
60. n karyotypic order The hg19 PAR regions are defined as follows Table 26 hg19 PAR regions Name Chr Start Stop PAR 1 X 60 001 2 699 520 PAR 2 X 154 931 044 155 260 560 PAR 1 Y 10 001 2 649 520 PAR 2 N 59 034 050 59 363 566 You can find links to Illumina iGenomes references here support illumina com sequencing sequencing software igenome ilmn y NOTE The version of hg19 provided in iGenomes is not PAR masked Whole Genome Sequencing Services User Guide P 5 s 1e19q oi 1oeds 91U0U959 Analysis Overview Isaac Aligner The Isaac Aligner aligns DNA sequencing data single or paired end with read lengths of 32 150 bp 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 1 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 Ilumina sequencing platforms Bioinformatics 29 16 2041 3 bioinformatics oxfordjourna
61. orrected based on known ambiguities in the reference flagged during candidate mapping Following alignment reads are sorted Further analysis is performed to identify duplicates and optionally to realign indels Alignment Output After sorting the reads the Isaac Aligner generates compressed binary alignment output files called BAM bam files using the following process 2 6 Part 15040892 Rev D 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 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 reads data from base call BCL files that were written during base calling on the sequencer to generate the read names Data are then compressed into blocks of 64 kb or less to create the BAM file Whole Genome Sequencing Services User Guide 2 J9UBIy oees Analysis Overview Isaac Variant Caller The Isaac Variant Caller identifies single nucleotide variants SNVs and small indels using the following steps Read filtering Filters out reads fa
62. ple These files along with the manifest cluster and genotyping product files can be imported into the Ilumina GenomeStudio software genotyping module www illumina com software genomestudio software ilmn to reproduce the genotyping calls The genotyping product files can be found on the Array support page in the Downloads tab To find the version of array chip used for your project refer to the sample sheet in each sample folder The sample sheet can be found in the following directory of each sample folder Sample_Barcode Genotyping Sample Barcode idats If available gtc files with genotype call files for use as input into genotyping software for reanalysis are also included Sample Barcode Genotyping vcf gz This file contains the genotyping SNPs in VCF format The genotyping SNPs were mapped to the reference using megaBLAST and filtered in the following manner Exclusions intensity only SNPs any match not aligning to the SNP any probe with a hamming distance greater than or equal to 5 any probe where the highest scoring mapping site is not the best matching site ie there is another site or sites within an identical hamming distance Any genotyping probe not matching the reference or excluded from the mapping will be mapped to chromosome NA in the vcf file The following fields are utilized in the vcf file Table 4 INFO Fields ID Description AL Array alleles relative to the design strand of the array probe ST The
63. ploid model and adjusted for the other copy number models For example if the mean u and standard deviation o are estimated to be 100 and 15 in the diploid model then corresponding estimates in the haploid model would be u 2 and 0 2 The mean and standard deviation are estimated using the autosomal median and MAD of counts This model is the default as it is more appropriate in cases where the spread of counts is higher than expected from the Poisson model due to unaccounted sources of variability An example of this case is single cell sequencing experiments where whole genome amplification is required Following assignment of copy number states neighboring regions that received the same copy number call are merged into a single region Phred scaled Q scores are assigned to each region using a simple logistic function derived using array CGH data as the gold standard The probability of a miscall is modeled as p 1 1 1 e 0 5532 0 147N Where N is the number of bins found within the nondiploid region This probability is converted to a Q score by q 10 log p This estimate is likely conservative as it is derived from array CGH Importantly Q scores are a function of number of bins not genomic size so they are applicable to experiments of any sequencing depth including low depth cytogenetics screening The coordinates of nondiploid regions and their Q scores are output to a VCF file Two filters are applied to PASS variants Fir
64. re found per edge 2 Analyze graph edges to find SVs The second step is to analyze individual graph edges or groups of highly connected edges to discover and score SVs associated with the edges These substeps of this process include Inference of SV candidates associated with the edge Attempted assembly of the SVs break ends Scoring and filtration of the SV under various biological models currently diploid germline and somatic Output to VCF Capabilities 40 Isaac SV Caller can detect all structural variant types that are identifiable in the absence of copy number analysis and large scale de novo assembly Detectable types are enumerated in this section For each structural variant and indel Isaac SV Caller attempts to align the break ends to base pair resolution and report the left shifted break end coordinate per the VCF 4 1 SV reporting guidelines Isaac SV Caller also reports any break end microhomology sequence and inserted sequence between the break ends Often the assembly fails to provide a confident explanation of the data In such cases the variant is reported as IMPRECISE and scored according to the paired end read evidence alone The sequencing reads provided as input to Isaac SV Caller are expected to be from a paired end sequencing assay that results in an inwards orientation between the 2 reads of each DNA fragment Each read presents a read from the outer edge of the fragment insert inward Part 15040892 Rev
65. s greater than 0 3 HighSNVSB site SNV strand bias value SNVSB exceeds 10 IndelConflict indel The locus is in region with conflicting indel calls IndelSizeFilter indel Indel is outside reportable size range Insertion Deletion range reported in VCF header LowGQX site indel Locus GQX is less than 30 or not present SiteConflict indel The site genotype conflicts with the proximal indel call This call is typically a heterozygous SNV call made inside a heterozygous deletion VCF Filter ID Type Description HAPLOID_ site indel Locus has heterozygous genotype in a haploid region CONFLICT HighDepth site indel The locus depth is greater than 3x the mean chromosome depth HighDPFRatio site The fraction of base calls filtered out at a site is greater than 0 3 HighSNVSB site SNV strand bias value SNVSB exceeds 10 IndelC onflict indel The locus is in region with conflicting indel calls IndelSizeFilter indel Indel is outside reportable size range Insertion Deletion range reported in VCF header LowGOX site indel Locus GOX is less than 30 or not present SiteConflict indel The site genotype conflicts with the proximal indel call This call is typically a heterozygous SNV call made inside a heterozygous deletion Part 15040892 Rev D Isaac Copy Number Variant Caller Isaac Copy Number Variant CNV Caller is an algorithm for calling copy number variants from a diploid sample Most of a normal DNA sample is diploid or having
66. saac CNV Caller and Isaac SV Caller The following output is produced Realigned and duplicate marked reads in a bam file format Variants in a 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 The reference calls are block compressed and all single nucleotide polymorphisms and indels are included Currently Structural Variants and CNVs are kept in separate files Figure 1 Whole Genome Sequencing Pipeline Alignment Variant Analysis Isaac Isaac Variant Caller gt BAY gt Small Variants Isaac Structural Variant Caller Isaac Copy Number Variant Caller 24 Data Output gt VCF SNV and small indels gVCF gt Reference and small variant sites gt VCF Structural Variants VCF Copy Number Variants Part 15040892 Rev D Genome Specific Details Illumina currently uses hg19 from UCSC as a reference genome The chromosome naming scheme follows the UCSC conventions of chr1 22 chrX chrY chrM The pseudoautosomal region PAR of the Y chromosome is masked out with N s The result of this is that any mappings occurring in the PAR region map to the X chromosome Currently only the main chromosomes and mitochondria are used in the reference none of the nonmapped contigs are included As per GATK specification for UCSC chrM is the first chromosome followed by the rest i
67. ses of the call are ignored Tf 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 26 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 Part 15040892 Rev D This method treats 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 SNV and indel genotyping methods are complete the variant caller applies a final set of heuristic filters to produce the final set of
68. st a variant must have a Q score of Q10 or greater Second a variant must be of size 10 kb or greater Whole Genome Sequencing Services User Guide 3 9 19 19 JUBUBA Jaquiny Ado peges Analysis Overview Isaac Structural Variant Caller Isaac Structural Variant SV Caller is a structural variant caller for short sequencing reads It can discover structural variants of any size and score these variants using both a diploid genotype model and a somatic model when separate tumor and normal samples are specified Structural variant discovery and scoring incorporate both paired read fragment spanning and split read evidence Method Overview Isaac SV Caller works by dividing the structural variant discovery process into 2 primary steps scanning the genome to find SV associated regions and analysis scoring and output of SVs found in such regions 1 Build SV association graph In this step the entire genome is scanned to discover evidence of possible SVs and large indels This evidence is enumerated into a graph with edges connecting all regions of the genome that have a possible SV association Edges can connect 2 different regions of the genome to represent evidence of a long range association or an edge can connect a region to itself to capture a local indel small SV association These associations are more general than a specific SV hypothesis in that many SV candidates can be found on 1 edge although typically only 1 or 2 candidates a
69. t only alignments that start at unique 35 mer positions in the genome are used Bin Sizes Isaac CNV Caller is initialized with 100 alignments per bin and then proceeds to compute the bin boundaries such that each bin contains the same bin size or number of unique 35 mers The term bin size refers to the number of unique genomic 35 mers per bin Because some regions of the human genome are more repetitive than others physical bin sizes in genomic coordinates are not identical In the following example each box is a position along the genome Each checkmark represents a unique 35 mer while each X represents a nonunique 35 mer The bin size in this example is 3 3 checkmarks per bin The physical size of each bin is not constant B1 and B3 have a physical size of 3 but B2 and B4 have physical sizes of 4 and 6 respectively Computing Bin Size To compute bin size the ratio of observed alignments to unique 35 mers is calculated for each autosome The desired number of alignments per bin is then divided by the median of these ratios to yield bin size For whole genome sequencing bin sizes are typically in the range of 800 1000 unique 35 mers Correspondingly most physical window sizes are in the 1 1 2 kb range The advantage of this approach relative to using fixed genomic intervals is that the same number of reads map to each bin regardless of uniqueness or ability to be mapped After bin size is computed bins are defined as
70. t remote translocation mate break end ID of mate break end Read pairs supporting this variant where both reads are confidently mapped Length of microinsertion at event breakpoints Sequence of microinsertion at event breakpoints Difference in length between REF and ALT alleles Type of structural variant described in the ALT field Reference sequence upstream of the variant Confidently mapped reads supporting this variant at the upstream break end it is possible that mapping is not confident at downstream break end ID Description GQ Genotype Quality GT Genotype PR Spanning paired read support for the REF and ALT alleles in the order listed SR Split reads for the REF and ALT alleles in the order listed for reads where P allele read gt 0 999 Sample_Barcode genome vcf gz The genome vcf file contains vcf formatted output for the SNVs indels and block compressed nonvariant position output You can use this file to compare variants and covered regions between samples quickly The filters and INFO fields are a combination of both the SNV and indel vcf files listed below along with the block compressed specific flags See Genome VCF gVCF on page 30 for details For additional INFO fields pertaining to annotation information see Introduction on page 24 Sample_Barcode vcf gz This VCF file contains SNV and indel calls along with basic annotations Nonvariant positions are not included 15
71. tBio 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 Read Before Using this Product This Product and its use and disposition is subject to the following terms and conditions If Purchaser does not agree to these terms and conditions then Purchaser is not authorized by Ilumina to use this Product and Purchaser must not use this Product 1 Definitions Application Specific IP means Ilumina owned or controlled intellectual property rights that pertain to this Product and use thereof only with regard to specific field s or specific application s Application Specific IP excludes all Ilumina owned or controlled intellectual property that cover aspects or features of this Product or use thereof that are common to this Product in all possible applications and all possible fields of use the Core IP Application Specific IP and Core IP are separate non overlapping subsets of all Ilumina owned or controlled intellectual property By way of non limiting example Ilumina intellectual property rights for specific diagnostic methods for specific forensic methods or for specific nucleic acid biomarkers sequences or combinations o
72. ta integrity you can use the md5sum tool to find the inconsistencies Use the tool to compare the hash from the provided md5sum file to one generated from the downloaded file On a Unix system you can use the following commands to perform an md5sum check assuming the utility is installed cd Sample_Barcode md5sum c md5sum txt The check verifies every file and require approximately 30 45 minutes to complete Any errors are listed in the output In Windows there are various command line and GUI tools available to perform an md5sum check The Cygwin tools provide a utility identical to Linux e A11163 u e1eq Analysis Overview INTO 5 ozi o q RREO SD SRD RD Lo RPC ho O 24 Genome Specific Detalls 25 EEE ya ect enced ste tarts eee oe essa nee Geena pe dns en eee adia atas 26 Isaac Variant Caller nn 28 Genome VOF 0VCF sas secucsadegends Doe ssa dos e des DAN rn ed 30 Isaac Copy Number Variant Caller cc cae ooo 35 Isaac Structural Variant Caller 40 a E pt 3 GE NOS K Y X Em E dd a to ean SE aa o E T E T rr e a varr Whole Genome Sequencing Services User Guide 2 3 C Jejdeuyo Analysis Overview Introduction After the sequencer generates base calls and quality scores the resulting data are analyzed in 2 steps alignment to the reference genome followed by assembly and variant calling Alignment and variant calling are performed with the Isaac Alignment Software Isaac Variant Caller I
73. tail The Isaac Variant Caller provides the following blocking scheme min30p3a as the nonvariant block compression scheme Each sample value shown for the block such as the depth using the DP key is restricted to have a range where the maximum value is within 30 or 3 of the minimum Therefore for sample value range x y y lt x max 3 x 0 3 This range restriction applies to all sample values written in the final block record Genotype Quality for Variant and Nonvariant Sites The gVCF file uses an adapted version of genotype quality for variant and nonvariant site filtration This value is associated with the GQX key The GQX value is intended to represent the minimum of Phred genotype quality assuming the site is variant assuming the sites is nonvariant You can use this value to allow a single value to be used as the primary quality filter for both variant and nonvariant 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 for example An assertion that a site is homozygous reference at GQX gt 30 is made assuming the site is variant An assertion that a site is a nonreference genotype at GQX 2 30 is made assuming the site is nonvariant Whole Genome Sequencing Services User Guide 3 3 14016 49A auous5 Analysis Overview Filter Criteria 34 The gVCF FILTER description is d
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76. ts that are necessary for Purchaser s intended uses of this Product including without limitation any rights from third parties or rights to Application Specific IP Illumina makes no guarantee or warranty that purchaser s specific intended uses will not infringe the intellectual property rights of a third party or Application Specific IP Part 15040892 Rev D 3 Regulatory This Product has not been approved cleared or licensed by the United States Food and Drug Administration or any other regulatory entity whether foreign or domestic for any specific intended use whether research commercial diagnostic or otherwise This Product is labeled For Research Use Only Purchaser must ensure it has any regulatory approvals that are necessary for Purchaser s intended uses of this Product 4 Unauthorized Uses Purchaser agrees a to use each Consumable only one time and b to use only Ilumina consumables reagents with Ilumina Hardware The limitations in a b do not apply if the Documentation or Specifications for this Product state otherwise Purchaser agrees not to nor authorize any third party to engage in any of the following activities i disassemble reverse engineer reverse compile or reverse assemble the Product ii separate extract or isolate components of this Product or subject this Product or components thereof to any analysis not expressly authorized in this Product s Documentation iii gain access to or attempt to determine
77. uch flags and using the including f or the excluding F option with flags from SAMtools you can filter extract any kind of read from the Bam Sam file The hexadecimal outputs are a bit hard to decipher To convert the SAMtools flags into a human readable format you can input the flag into picard sourceforge net explain flags html or run the following command to output the flags in the coded string format described in the samtools manual Ssamtools view X Example bam A few commonly used examples of filtering on flags are detailed below Extract all reads that are unmapped f 4 include reads which are unmapped command will output all the reads which are not mapped Ssamtools view h f 4 Example bam Extract reads with unmapped mates f 8 include reads whose mates are not mapped command will output all reads whose mates are not mapped Ssamtools view h f 8 Example bam Extract an unmapped read with a mapped mate f 4 include reads which are unmapped F 8 exclude reads whose mate is not mapped command outputs reads that are unmapped with the corresponding mate mapped Ssamtools view h f 4 F8 Example bam Extract a mapped read with an unmapped mate f 8 include reads whose mate is unmapped F 8 exclude all reads not mapped command outputs reads which are mapped with the mate is unmapped Ssamtools view h f 8 F4 Example bam Extract both reads of a pair which are unmapped
78. umber Ajquessy cluster id Unpadded 0 based cluster id in the order in which the clusters appear In cases where the x y coordinates from the flow cell were preserved this column will contain the y coordinate while the cluster id will contain the x coordinate Otherwise this will always contain 0 lane number Lane number 1 8 tile number Unpadded tile number Bitwise Flag Notes FLAG The bitwise flags used are as follows Table 2 Bitwise Flags Bit Description 0x1 Template having multiple segments in sequencing 0x2 Each segment properly aligned according to the aligner 0x4 Segment unmapped 0x8 Next segment in the template unmapped 0x10 SEQ being reverse complemented 0x20 SEO of the next segment in the template being reversed 0x40 The first segment in the template 0x80 The last segment in the template 0x100 Secondary alignment 0x200 Not passing quality controls 0x400 PCR or optical duplicate Extended Tags and Optional Fields Note Always set on for paired reads Pair matches dominant template orientation Set for unmapped reads Paired read is unmapped Read mapped to strand of reference Paired read mapped to strand of reference Read 1 sequence Read 2 sequence Isaac does not produce secondary alignments Nonpass filter reads are not included always off Read 1 and Read 2 were marked as duplicate reads The aligner produces the following

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