Genome in a Bottle Consortium 2013: standards for validating high-throughput variant detection

These are my notes from the 2013 Genome in a Bottle Consortium meeting, focused around developing reference materials and standards for measuring variation detection for human genome sequencing. The consortium is a fully open community that seeks to expand its scope to include additional materials and standards within the general goal of being able to accurately characterize how well a sequencing analysis performs. Marc Salit provided a nice introductory overview of these goals and presented an open invitation to define the scope and plans for the consortium.


Justin Zook described his work on developing the current Genome in a Bottle reference materials: an integrated call set for the well-sequenced NA12878 genome. The goal is to define genotypes with high confidence, including distinguishing homozygous reference and uncertain calls to assess both false positives as well as negatives. It’s a stringent set of calls integrating 12 different inputs and after stringent filtering has 2.3 million variants covering 82.3% of the genome. The reference calls are available on the GCAT variant comparison platform for comparison. One current difficulty with comparing high quality datasets is different variant representations: important to normalize these.

Michael Eberle from Illumina talked about their Platinum Genome initiative. Goal is to develop a catalog of known mutations and toolkit to compare algorithms to it. Using a pedigree analysis on 17 individuals in the NA12878 family. The family structure allows generation of haplotypes, allowing identification of errors. Use calls from GATK, cortexvar, Isaac and complete genomics. Identify CNVs using BreakDancer and Grouper. After initial predictions, overlap between methods and corroborate based on read counts. Of the confident 772 total events identified by this method: 408 called by both, 266 by BreakDancer, 98 by Grouper. Another idea is to look at variants in these regions since you expect loss of heterozygosity in deleted regions. Found more deletions than amplifications in CNVs. Biggest current gap is in events from 30bp to 2kb.

Francisco De La Vega from Real Time Genomics discussed approaches to using trios to help develop reference materials. By assessing Mendelian inheritance from the full family using Bayesian priors for parents and children, improve calling. By specifically investigating relationships, this does better than standard family based calling, ala GATK populations. Got 150k more calls than GATK using this approach. Has 94.1% sensitivity in GiaB regions compared to 92.5% for GATK. Reduces mendelian inconsistency dramatically, which makes good sense since the approach optimizes for that.

Deanna Church discussed the GeT-RM project from NCBI and CDC. GeT-RM incorporates data from 12 labs. Integration is a pain and have to deal with each input format specifically. They have a nice browser that allows viewing the calls in-line with the BAM files. This helps identify discordant variants and potential causes from specific submissions. Identified regions verified from multiple submissions but with no alignment evidence. Website provides high quality variants in VCF format and BED file of Sanger verified regions. Plans going forward involve development of additional web tools, including incorporation with new genome reference: GRCh38 is coming soon. A lot of realignment is coming as well.

Working groups

Reference Materials Selection

In progress cancer calling assessment work: NCI Cancer spike in, TGen-Illumina tumor-normal pairs, HorizonDX cell lines. Jason Lih from NCI has a set of plasmid constructs with engineered mutations relative to cancer. Sanger confirmed sequences. Stephanie Pond from Illumina collaborating with TGen on COLO829B/COLO829: has 37 confirmed mutations. Also have 9 synthetic fusion constructs spiked into COLO829. Jonathan Frampton from Horizon Diagnostics has engineered cancer mutations in 3 cancer cell lines. Lots of nice setups for replicating FFPE and dilution series. Have ~36 additional mutations. Kara Norman from AcroMetrix discussed engineered cell line controls with 12-26 engineered variations from COSMIC.

Reference Material Characterization

General goal: get as much data as possible. Would like to be able to apply Moleculo to NA12878 to get phasing information. Also add in 20-30x PacBio data, Optimal mapping from BioNanoGenomics, 454 and Ion. For PGP trio data, plan to characterize with Illumina, Complete, PacBio and Moleculo. Overall should help with phasing completeness and help resolve conflicts.

Bioinformatics working group

Reviewed the data release policy, which follows the Fort Lauderdale approach: can get data but can’t publish on it until the consortium does. Existing resources for NA12878: NIST, X Prize, Illumina Platinum Genomes, 1000 genomes, Real Time Genomics, Broad chr20 and exome. Personalis has CNV calls for NA12878.

For data integration, trying to use pedigree methods and multi-platform methods together. Critical issue is handling growth in datasets. Emphasize importance of recognizing heterogeneity across the genome, which the performance metrics group handles. Quality of variants is difficult to assess since not calibrated across multiple approaches: need a way to map the quality onto a standard scale during data integration.

Data from NA12878 and parents NA12891/NA12892 available from FTP/Aspera site at NCBI and a mirrored giab Amazon S3 bucket.

Goal is to develop automated workflows for future reference materials so we can handle multiple new genomes including PGP data.

Performance metrics

  • Metrics to measure
    • Laboratory side: library prep, platform, lots of other metrics
    • How do you motivate to make this worth while? Hard problem to capture and upload.
    • Other issues: lots of variables with lots of incompleteness, tricky statistics.
    • Decisions: flattened key/value JSON structure for metadata. Circulate list of things to provide.
  • Provide better characterization of trusted/non-trusted genome regions with provided BED files of:
    • Trusted regions: robust across multiple technologies and protocols
    • Less trusted regions: difficult to call in regions that might be do-able on specific technologies or approaches but are not as reliable.
    • Non called regions: Regions we cannot characterize.
  • Development of infrastructure to assess and report accuracy. General goal is to merge existing software tools.
    • Get-RM
    • X Prize/Harvard School of Public Health: bcbio.variation handles comparison of variant calls to reference genomes and reporting of concordant/discordants
    • Harvard School of Public Health: bcbio-nextgen pipeline for variant calling and comparison
    • Chris Mason’s software (name?)


One thought on “Genome in a Bottle Consortium 2013: standards for validating high-throughput variant detection

  1. Pingback: Friday SNPpets | The OpenHelix Blog

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