Variant (SNPs/Indels) calling in DNA sequences, Part 2

[www.absolutefab.com]
Variant calling for disease association (2/2)
Searching the haystack
July 14, 2011

Quick recap: DNA sequence read mapping
July 14, 2011
Sequencing->FASTQ->alignment to reference genome
Resulting file type: BAM
Visualized in Genome Viewer
“What genomic regions were sequenced?”
Quality Control
Projects
Fastq
Bam

Production Informatics and Bioinformatics
July 14, 2011
Produce raw sequence reads
Basic Production
Informatics
Map to genome and generate raw genomic features (e.g. SNPs)
Advanced
Production Inform.
Analyze the data; Uncover the biological meaning
Bioinformatics
Research
Per one-flowcell project

Good mapping is crucial
Mapping tools compromise accuracy for speed: approximate mapping.
Identifying exactly where the reads map is the fundament for all subsequent analyses.
The exact alignment of each read is especially important for variant calling.
July 14, 2011
by neilalderney123

Mapping challenges
Incorrect mapping
Amongst 3 billion bp (human) a 100-mer can occur by chance
Multi-mappers
The genome has none-unique regions (e.g. repeats) one read mapping to multiple sites can happen
Duplicates
PCR duplicates can introduce artifacts.
July 14, 2011
Streptococcus suis (squares)
Musmusculus (triangles)
ACGATATTACACGTACACTCAAGTCGTTCGGAACCT
TTACACGTACA
TACACGTACAC
ACACGTACACT
CACGTACTCTC
CACGTACTCTC
CACGTACTCTC
CACGTACTCTC
Turner DJ, Keane TM, Sudbery I, Adams DJ. Next-generation sequencing of vertebrate experimental organisms. Mamm Genome. 2009 Jun;20(6):327-38. PMID: 19452216

Methods for ensuring a good alignment
Biological: Using paired end reads to increase coverage
Bioinformatically:
Local-realignment
Base pair quality score re-calibration
July 14, 2011
~200 bp
?
Repeat region

Local Realignment (GATK)
July 14, 2011
QBI data
Local realignment of all reads at a specific location simultaneously to minimize mismatches to the reference genome
Reduces erroneous SNPs refines location of INDELS
original
realigned
DePristo MA, et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet. 2011 May;43(5):491-8. PMID: 21478889

Quality score recalibration (GATK)
PHRED scores are predicted
Looking at all reads at a specific location allows a better estimate on base pair quality score.
Excludes all known dbSNP sites
 Assume all other mismatches are sequencing errors
Compute a new calibration table bases on mismatch rates per position on the read
Important for variant calling
July 14, 2011
Thomas Keane 9th European Conference on Computational Biology 26th September, 2010

Recalibration of quality score
July 14, 2011
All bases are called with Q25
In reality not all are that good:
bases actually mismatch the reference at a 1 in 100 rate, so are actually Q20” GATK

Variant calling methods
> 15 different algorithm
Three categories
Allele counting
Probabilistic methods, e.g. Bayesian model
to quantify statistical uncertainty
Assign priors e.g. by taking the observed allele frequency of multiple samples into account
Incorporating linkage disequilibrium (LD)
Specifically helpful for low coverage and common variants
July 14, 2011
variant
SNP
Ref
A
Ind1
G/G
Ind2
A/G
Nielsen R, Paul JS, Albrechtsen A, Song YS. Genotype and SNP calling from next-generation sequencing data. Nat Rev Genet. 2011 Jun;12(6):443-51. PMID: 21587300.
http://seqanswers.com/wiki/Software/list

VCF format
[HEADER LINES]
#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT NA12878
chr1 873762 . T G 5231.78 PASS [ANNOTATIONS] GT:AD:DP:GQ:PL 0/1:173,141:282:99:255,0,255
chr1 877664 rs3828047 A G 3931.66 PASS [ANNOTATIONS] GT:AD:DP:GQ:PL 1/1:0,105:94:99:255,255,0
chr1 899282 rs28548431 C T 71.77 PASS [ANNOTATIONS] GT:AD:DP:GQ:PL 0/1:1,3:4:25.92:103,0,26
chr1 974165 rs9442391 T C 29.84 LowQual [ANNOTATIONS] GT:AD:DP:GQ:PL 0/1:14,4:14:60.91:61,0,255
Individual statistics
GT - genotype - 0/1
AD – total number of REF/ALT seen – 173 T, 141 A
DP – depth MAPQ > 17 – 282
GQ - Genotype Quality - 99
PL – genotype likelihood - 0/0: 10-25.5=unlikely, 0/1:10-0=likely, and 1/110-25.5=unlikely
Location statistics, e.g.
Strand bias
How many reads have a deletion at this site
July 14, 2011

When to call a variant ?
July 14, 2011
Hom
REF: 0%
ALT: 100%
Het
REF: 50%
ALT: 50%
??
REF: 77%
ALT: 23%
QBI data
QBI data

Hard Filtering
Reducing false positives by e.g. requiring
Sufficient Depth
Variant to be in >30% reads
High quality
Strand balance
…
Subjective and dangerous in this high dimensional search space
July 14, 2011
Strand Bias
Bentley, D.R. et al. Accurate whole human genome sequencing using reversible terminator chemistry. Nature 456, 53–59 (2008).
Wheeler, D.A. et al. The complete genome of an individual by massively parallel DNA sequencing. Nature 452, 872–876 (2008).
QBI data

Gaussian mixture model
Train on trusted variants and require the new variants to live in the same hyperspace
Potential problem: Overfitting and biasing to features of knownSNPs!!!
July 14, 2011

Indel calling
First local realignment might not be sufficient to confidently determine the beginning and end of indels
Dindel-algorithm
Local realignment for every indel candidate
July 14, 2011
Albers CA, Lunter G, Macarthur DG, McVean G, Ouwehand WH, Durbin R. Dindel: Accurate indel calls from short-read data. Genome Res. 2011 Jun;21(6):961-73. PMID: 20980555.

Recap
July 14, 2011

Outcome: How many variants will I find ?
July 14, 2011
Hiseq: whole genome; mean coverage 60; 101PE; (NA12878)
Exome: agilent capture; mean coverage 20; 76/101PE; (NA12878)

Three things to remember
Getting the mapping right is critical
Variant calling is not merely to count the differences
Just listing the variants does not tell you anything biologically relevant.
July 14, 2011
by Яick Harris
Fernald GH, Capriotti E, Daneshjou R, Karczewski KJ, Altman RB. Bioinformatics challenges for personalized medicine. Bioinformatics. 2011 Jul 1;27(13):1741-8. PMID: 21596790

Next week:
July 14, 2011
Abstract: This seminar aims at answering the question of what to make of the identified variants, specifically how to evaluate the quality, prioritize and functionally annotate the variants.

Walk-in-clinic
July 14, 2011

Variant (SNPs/Indels) calling in DNA sequences, Part 2

by Denis Bauer on Jul 13, 2011

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Variant (SNPs/Indels) calling in DNA sequences, Part 2 — Presentation Transcript