sept2016 smallvar nist intro
TRANSCRIPT
Genome in a Bottle WorkshopSmall Variant Data Jamboree
Justin Zook and Marc SalitNIST Genome-Scale Measurements
Group
September 15, 2016
Integration Methods to Establish Reference Variant Calls
Candidate variants
Concordant variants
Find characteristics of bias
Arbitrate using evidence of bias
Confidence Level Zook et al., Nature Biotechnology, 2014.
Integration Methods to Establish Reference Variant Calls
Candidate variants
Concordant variants
Find characteristics of bias
Arbitrate using evidence of bias
Confidence Level Zook et al., Nature Biotechnology, 2014.
NEW: Reproducible
integration pipeline
with new calls for
NA12878 and PGP
Trios!
New calls (v3.3) vs. old calls (v2.19)
V3.3• 3441361 match PG• 550982 PG calls outside
high conf• 124715 calls not in PG• After excluding low
confidence regions and regions around filtered PG calls:– 40 calls not in PG– 60 extra PG calls
V2.19 • 3030717 match PG• 1018795 PG calls outside
high conf• 122359 calls not in PG• After excluding low
confidence regions and regions around filtered PG calls:– 87 calls not in PG– 404 extra PG calls
New calls (v3.3) vs. old calls (v2.19)
V3.3• 3441361 match PG• 550982 PG calls outside
high conf• 124715 calls not in PG• After excluding low
confidence regions and regions around filtered PG calls:– 40 calls not in PG– 60 extra PG calls
V2.19 • 3030717 match PG• 1018795 PG calls outside
high conf• 122359 calls not in PG• After excluding low
confidence regions and regions around filtered PG calls:– 87 calls not in PG– 404 extra PG calls
More high-confidence calls match Platinum Genomes
New calls (v3.3) vs. old calls (v2.19)
V3.3• 3441361 match PG• 550982 PG calls outside
high conf• 124715 calls not in PG• After excluding low
confidence regions and regions around filtered PG calls:– 40 calls not in PG– 60 extra PG calls
V2.19 • 3030717 match PG• 1018795 PG calls outside
high conf• 122359 calls not in PG• After excluding low
confidence regions and regions around filtered PG calls:– 87 calls not in PG– 404 extra PG calls
Similar extra calls not in Platinum Genomes
New calls (v3.3) vs. old calls (v2.19)
V3.3• 3441361 match PG• 550982 PG calls outside
high conf• 124715 calls not in PG• After excluding low
confidence regions and regions around filtered PG calls:– 40 calls not in PG– 60 extra PG calls
V2.19 • 3030717 match PG• 1018795 PG calls outside
high conf• 122359 calls not in PG• After excluding low
confidence regions and regions around filtered PG calls:– 87 calls not in PG– 404 extra PG calls
~80% fewer differences from PG in high confidence regions
New calls (v3.3) vs. old calls (v2.19)Example vcf (verily) Stratified
V3.3• 17% of SNPs not assessed
– 23% of SNPs in RefSeq coding– 53% of SNPs in “bad
promoters”• 78% of indels not assessed
– 0.7% difference rate• 17% FP in regions
homologous to decoy
V2.19 • 27% of SNPs not assessed
– 36% of SNPs in RefSeq coding– 82% of SNPs in “bad
promoters”• 78% of indels not assessed
– 1.2% difference rate• 0.2% FP in regions
homologous to decoy
Principles of Integration Process
• Form sensitive variant calls from each dataset
• Define “callable regions” for each callset
• Filter calls from each method with annotations unlike concordant calls
• Compare high-confidence calls to other callsets and manually inspect subset of differences– vs. pedigree-based calls– vs. common pipelines– Trio analysis
• When benchmarking a new callset against ours, most putative FPs/FNs should actually be FPs/FNs
Criteria for including new callsets• Form sensitive variant
calls from each dataset• Define “callable regions”
for each callset• Good coverage and MapQ• Use knowledge about
technology and manual inspection to exclude repetitive regions difficult for each dataset
• For new callsets, ensure most FNs in callable regions relative to current high-confidence calls are questionable in the current calls
• Filter calls from each method with annotations unlike concordant calls– Annotations for which
outliers are expected to indicate bias should be selected for each callset
Ongoing work: With sufficient coverage, 10X phasing seems to specifically identify most SNP
errors identified by pedigree phasing
Collaboration with Nathan Edwards and
Zhezhen Wang at Georgetown
Univ
Ongoing work: How can we add more complex events that are not normalized?• Current integration only
breaks into primitives– Some complex calls end
up uncertain– If part of a complex
variant is uncertain, we exclude the whole region
• 3 approaches– Kevin Jacobs vgraph
• Merge all callsets into a single graph
• Still need to work on partial complex calls
– Chen Sun and Paul Medvedev – varmatch• Start with one callset and match
otther callers one at a time, adding in new variants from each
– Sean Irvine and Len Trigg, RTG – vcfeval• Presentation today
Ongoing work: GRCh38
• Draft calls for chr20 on GRCh38
• Make calls on mapped reads for Illumina and 10X
• Lift over calls for CG, Ion, and SOLiD
• Preliminary comparisons to PG seem similar to those for GRCh37
Ongoing/Future Work and Questions
• Integrate with pedigree calls for NA12878– Mike Eberle, Illumina
• Integrating phasing information from family, linked reads, etc.– Sean Irvine/Len Trigg, RTG
• Integrate complex variants– Sean Irvine/Len Trigg, RTG– Chen Sun/Paul Medvedev,
PSU
• Incorporate more calls in difficult-to-map regions– 10X– Dovetail– PacBio
• How to integrate indels 15-50bp?
• Using ALT loci
Acknowledgements
• NIST– Marc Salit– Jenny McDaniel– Lindsay Vang– David Catoe
• Genome in a Bottle Consortium
• GA4GH Benchmarking Team
• FDA– Liz Mansfield– Zivana Tevak– David Litwack