1.
Genome in a Bottle: Integrating human sequence data sets provides
a resource of benchmark SNP and indel genotype calls
Justin
1,
Zook
Brad
2,
Chapman
Oliver
2,
Hofmann
Winston
2,
Hide
Jason
3,
Wang
David
3,
Mittelman
1National
Institute of Standards and Technology, Gaithersburg, MD
2Harvard School of Public Health, Cambridge, MA; 3Arpeggi, Inc., Austin, TX
1
Integrating SNPs & indels
Genome in a Bottle
Consortium
• As sequencing moves to clinical
applications, assessing accuracy
becomes very important.
• With the Genome in a Bottle
Consortium, NIST is developing
methods to characterize whole
genome Reference Materials that
can be used to assess the
performance of whole genome
sequencing
Samples
Spike-ins
Sample
Preparation
Unified
Genotyper
Force calls
with Unified
Genotyper
• Data from multiple sequencing
platforms and runs can be used to
understand and compensate for
errors and biases of each method
Force de novo
assembly with
Haplotype Caller
…
Unified
Genotyper
Haplotype
Caller
Force calls
with Unified
Genotyper
…
Force de novo
assembly with
Haplotype Caller
NA12878 Data sets
•
•
www.bioplanet.com/gcat
Interactive comparison of bioinformatics
methods to our integrated calls
• Using microarrays to assess
performance underestimates FN rate
•
Integrated calls have >20x higher percentage
of low complexity regions than microarrays
SNPs
indels
Find high-confidence SNP & indel sites
HomRef
SNP
VQSR
HomRef
indel
VQSR
HomVar
SNP
VQSR
HomVar
indel
VQSR
Het
indel
VQSR
…
HomRef
SNP
VQSR
Het SNP
VQSR
HomRef
indel
VQSR
HomVar
SNP
VQSR
HomVar
indel
VQSR
Het
indel
VQSR
Arbitrate using characteristics of mapping and
alignment bias and systematic sequencing
errors to find consensus SNP & indel sites
Indels/Complex Variants
Filter sites if <2 datasets are free of bias
• Multiple correct
representations of
complex variants
often exist
• Comparing complex CAGTGA > TCTCT complex variant
variants is difficult. Try RTG’s vcfeval!
Characteristics of bias
used for arbitration
•
•
• We propose a method using 14
datasets for CEPH/HapMap sample
NA12878 to find characteristics of
highly confident genotype calls and
use these characteristics to arbitrate
between discordant calls
Performance assessment
using integrated calls
• Freebayes has significantly improved
its indel calls over the past year:
Integrate UG
and HC calls for
dataset #11
• Systematic sequencing errors (SSEs)
Overlap of SNP calls for NA12878 between three variant call files.
(a) The three variant calls come from: (1) Illumina HiSeq reads mapped with bwa and
with variants called by GATK; (2) the same Illumina HiSeq reads mapped with bwa but
with variants called by samtools; (3) Complete Genomics called with CGTools 2.0.
(b) The samtools calls are replaced by SOLiD 4 reads called with GATK.
The gray numbers in parentheses are the numbers of variants that are not filtered in
the other datasets.
Genome in a
Bottle
Consortium
• Calls hosted on GCAT website
Haplotype
Caller
Integrate UG
and HC calls
for dataset #1
Sequencing
Variant list,
Performance
metrics
Cortex
Dataset #14
Candidate SNP & indel sites
Het SNP
VQSR
Bioinformatics
…
…
Dataset #1
Marc Salit1
Strand bias
Base Quality Rank Sum
• Local Alignment
•
•
•
•
•
• Mapping problems
•
•
•
Complete
Genomics
Distance from end of read
Mean position within read
Read Position Rank Sum
HaplotypeScore
Length of aligned reads
Illumina
HiSeq
Mapping Quality
Abnormal coverage – CNV
Length of aligned reads
• Abnormal allele balance
•
•
Allele Balance
Quality/Depth
Performance Assessment
• Within “highly confident” regions, all
datasets are highly sensitive and
specific
• Most “false” positives and negatives
appear to be microarray errors
Pedigree Methods
• Real Time Genomics and Illumina
Platinum Genomes have developed
methods to use the 11 children of
NA12878
• High-confidence variants are in
haplotypes that are properly
inherited in the children
Structural Variants
• Can we use similar methods for SVs?
• Arbitrate using coverage, insert
size, discordant paired
ends, mapping quality, softclipping, heterozygous/homozygous
ratio, allele fraction, …
• How to use long-read technologies?
Discussion
a http://genomeinabottle.org/blog-entry/existing-and-future-na12878-datasets.
• Genome in a Bottle Consortium
• New members welcome!
• www.genomeinabottle.org