INTRODUCTION TO NGS VARIANT CALLING ANALYSIS

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1 Hospital Universitari Vall d Hebron Institut de Recerca - VHIR Institut d Investigació Sanitària de l Instituto de Salud Carlos III (ISCIII) INTRODUCTION TO NGS VARIANT CALLING ANALYSIS Bioinformàtica per la Recerca Biomèdica Ferran Briansó 15/05/201

2 PRESENTATION OUTLINE 1 1. NGS WORKFLOW OVERVIEW 2 2. WET LAB STEPS 3 3. IMPORTANT SEQUENCING CONCEPTS. NGS ANALYSIS WORKFLOW 5 1. Primary analysis: de-multiplexing, QC 2. Secondary analysis: read mapping and variant calling 3. Tertiary analysis: annotation, filtering VISUALIZATION 6 6. COMMON PIPELINES AND FORMATS 7 7. CONCLUSIONS

3 1 NGS WORKFLOW OVERVIEW Extracted from Dr Kassahn's publicly shared slides (2013) 3

4 2 LIBRARY PREPARATION Select target Hybridization-based cature or PCR Add adapters Contain binding sequences Barcodes Primer sequences Amplify material

5 2 LIBRARY PREPARATION Select target Hybridization-based cature or PCR Add adapters Contain binding sequences Barcodes Primer sequences Amplify material A) Fragment DNA B) End-repair C) A-tailing, adapter ligation and PCR D) Final library contains sample insert indices (barcodes) flowcell binding sequences primer binding sequences 5

6 2 LIBRARY PREPARATION Select target Hybridization-based cature or PCR Add adapters Contain binding sequences Barcodes Primer sequences Amplify material 6

7 2 TEMPLATE PREPARATION Attachment of library e.g. To Illumina Flowcell Amplification of library molecules e.g. Brigde amplification 7

8 2 BRIDGE AMPLIFICATION 8

9 2 SEQUENCING Sequencing-by-Synthesis Detection by: Illumina fluorescence Ion Torrent ph ROCHE 5 PO and light 9

10 2 SEQUENCING-BY-SYNTHESIS (ILLUMINA) 10

11 1 3 IMPORTANT SEQUENCING CONCEPTS Barcoding/Indexing: allows multiplexing of different samples Single-end vs paired-end sequencing Coverage: avg. number reads per target Quality scores (Qscore): log-scales! 11

12 NGS DATA ANALYSIS WORKFLOW 12

13 DE-MULTIPLEXING (BARCODE SPLITTING) 13

14 FASTQ FORMAT see en.wikipedia.org/wiki/fastq_format 1

15 SEQUENCE QUALITY: fastqc Details of the output https://docs.google.com/document/pub?id=16gwpmwyw7o_r-zugcu8-osbby1gc97tfttingdk98ws 15

16 NGS DATA ANALYSIS WORKFLOW 16

17 READ MAPPING (BASIC ALIGNMENT) Comparison against reference genome (! not assembly!) Many aligners (short reads, longer reads, RNAseq...) Examples: BWA, Bowtie SAM/BAM files 17

18 BURROWS-WHEELER ALIGNMENT TOOL (BWA) Popular tool for genomic sequence data (not RNASeq!) Li and Durbin 2009 Bioinformatics Challenge: compare billion of short sequence reads (.fastq file) against human genome (3Gb) Burrows-Wheeler Transform to index the human genome and allow memory-efficient and fast string matching between sequence read and reference genome Li & Durbin 2009 Bionformatics 18

19 SAM/BAM FILES see 19

20 SAM/BAM Header (information regarding reference genome, alignment method...) 1) Read ID (QNAME) 2) Bitwise FLAG (first/second read in pair, both reads mapped...) 3) ReferenceSequence Name (RNAME) ) Position (POS, coordinate) 5) MapQuality (MAPQ = -10log10P[wrong mapping position]) 6) CIGAR (describes alignment matches, skipped regions, insertions..) 7) ReferenceSequence (RNEXT, Ref seq of the pair) 8) Position of the pair (PNEXT) 9) TemplateLength (TLEN) 10) ReadSequence 11) QUAL (in Fastq format, '*' if NA)... 20

21 VARIANT CALLING Identify sequence variants Distinguish signal vs noise VCF files Examples: SAMtools, SNVmix 21

22 SEQUENCE VARIANTS Differences to the reference 22

23 SEQUENCE VARIANTS Sanger: is it real?? NGS: read count Provides confidence (statistics!) Sensitivity tune-able parameter (dependent on coverage) 23

24 VARIANT CALLING: GATK Genome Analysis Toolkit (BROAD Institute) Initially developed for 1000 Genomes Project Single or multiple sample analysis (cohort) Popular tool for germline variant calling Evaluates probability of genotype given read data see and McKenna et al. Genome Research

25 SOMATIC VARIANT CALLING Somatic mutations can occur at low freq. (<10%) due to: Tumor heterogeneity (multiple clones) Low tumor purity (% normal cells in tumor sample) Requires different thresholds than germline variant calling when evaluating signal vs noise Trade-off between sensitivity (ability to detect mutation) and specificity (rate of false positives) Nature Reviews Cancer 12, (May 2012) 25

26 1 INDELS DETECTION Small insertions/ deletions The trouble with mapping approaches modified from Heng Li (Broad Institute) 26

27 INDELS DETECTION Small insertions/ deletions The trouble with mapping approaches 27

28 INDELS DETECTION Small insertions/ deletions The trouble with mapping approaches 28

29 RE-ALIGNMENT Re-align considering multi-read context, SNPs & INDELS previous info... adapted from Andreas Schreiber 29

30 EVALUATING VARIANT QUALITY TAKING INTO ACCOUNT: Coverage at position Number independent reads supporting variant Observed allele fraction vs expected (somatic / germline) Strand bias Base qualities at variant position Mapping qualities of reads supporting variant Variant position within reads (near ends or at centre) 30

31 VCF FILES Variant Call Format Standard for reporting variants from NGS Describes metadata of analysis and variant calls Text file format (open in Text Editor or Excel)!!! Not a MS Office vcard!!! see -version-1 31

32 VCF FILES 32

33 NGS DATA ANALYSIS WORKFLOW 33

34 VARIANT ANNOTATION Provide biological & clinical context Identify disease-causing mutations (among 1000s of variants) 3

35 ANNOTATION OVERVIEW 35

36 VARIANT FILTERING AND PRIORIZATION COMMON STEPS: PURPOSE: Identify pathogenic or disease-associated mutation(s) Reduce candidate variants to reportable set Remove poor quality variant calls Remove common polymorphisms Prioritize variants with high functional impact Compare against known disease genes Consider mode of inheritance (autosomal recessive, X-linked...) Consider segregation in family (where multiple samples available) 36

37 5 NGS DATA ANALYSIS WORKFLOW 37

38 5 VISUALIZATION IGV (or Genome Browser, Circos...) provided by Katherine Pillman 38

39 6 COMMON PIPELINE bcl2fastq (Illumina) FastQC (open-source) Exomes (HiSeq): BWA(open-source), GATK (Broad) Gene panels (MiSeq, PGM): MiSeq Reporter (Illumina) Torrent Suite (Ion Torrent) Custom scripts and third party tools (Annovar, snpeff, PolyPhen, SIFT...) Commercial annotation software (GeneticistAssistant, VariantStudio...) 39

40 6 COMMON DATA FORMATS.bcl.fastq.BAM.VCF.csv.txt.xls.html... 0

41 7 CONCLUSIONS NGS data - the new currency of (molecular) biology Broad applications (ecology, evolution, ag sciences, medical research and clinical diagnostics...). Rapidly evolving (sequencing technologies, library preparation methods, analysis approaches, software). Different tools/pipelines/parametrization gives different results, (more standards needed). Bioinformatics pipelines typically combine vendor software, third-party tools and custom scripts. Requires skills in scripting, Linux/Unix, HPC. Requires advanced hardware (not always available). Understanding of data (SE, PE, RNA-Seq) important for successful analysis. 1

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