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Archive for the ‘variation’ Category

Validating generalized incremental joint variant calling with GATK HaplotypeCaller, FreeBayes, Platypus and samtools

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Incremental joint variant calling

Variant calling in large populations is challenging due to the difficulty in providing a consistent set of calls at all possible variable positions. A finalized set of calls from a large population should distinguish reference calls, without a variant, from no calls, positions without enough read support to make a call. Calling algorithms should also be able to make use of information from other samples in the population to improve sensitivity and precision.

There are two issues with trying to provide complete combined call sets. First, it is computationally expensive to call a large number of samples simultaneously. Second, adding any new samples to a callset requires repeating this expensive computation. This N+1 problem highlights the inflexibility around simultaneous pooled calling of populations.

The GATK team’s recent 3.x release has a solution to these issues: Incremental joint variant discovery. The approach calls samples independently but produces a genomic VCF (gVCF) output for each individual that contains probability information for both variants and reference calls at non-variant positions. The genotyping step combines these individual gVCF files, making use of the information from the independent samples to produce a final callset.

We added GATK incremental joint calling to bcbio-nextgen along with a generalized implementation that performs joint calling with other variant callers. Practically, bcbio now supports this approach with four variant callers:

  • GATK HaplotypeCaller (3.2-2) – Follows current GATK recommended best practices for calling, with Variant Quality Score Recalibration used on whole genome and large population callsets. This uses individual sample gVCFs as inputs to joint calling.
  • FreeBayes (0.9.14-15) – A haplotype-based caller from Erik Garrison in Gabor Marth’s lab.
  • Platypus ( – A recently published haplotype-based variant caller from Andy Rimmer at the Wellcome Trust Centre for Human Genomics.
  • samtools (1.0) – The recently released version of samtools and bcftools with a new multiallelic calling method. John Marshall, Petr Danecek, James Bonfield and Martin Pollard at Sanger have continued samtools development from Heng Li’s code base.

The implementation includes integrated validation against the Genome in a Bottle NA12878 reference standard, allowing comparisons between joint calling, multi-sample pooled calling and single sample calling. Sensitivity and precision for joint calling is comparable to pooled calling, suggesting we should optimize design of variant processing to cater towards individual calling and subsequent finalization of calls, rather than pooling. Generalized joint calling enables combining multiple sets of calls under an identical processing framework, which will be important as we seek to integrate large publicly available populations to extract biological signal in complex multi-gene diseases.


There is not a consistent set of terminology around combined variant calling, but to develop one, here is how I’ll use the terms:

  • Joint calling – Calling a group of samples together with algorithms that do not need simultaneous access to all population BAM files. GATK’s incremental joint calling uses gVCF intermediates. Our generalized implementation performs recalling using individual BAMs supplemented with a combined VCF file of variants called in all samples.
  • Pooled or batch calling – Traditional grouped sample calling, where algorithms make use of read data from all BAM files of a group. This scales to smaller batches of samples.
  • Single sample calling – Variant calling with a single sample only, not making use of information from other samples.
  • Squaring off or Backfilling – Creating a VCF file from a group of samples that distinguishes reference from no-call at every position called as a variant in one of the samples. With a squared off VCF, we can use the sample matrix to consider call rate at any position. Large populations called in smaller batches will not be able to distinguish reference from no-call at variants unique to each sub-pool, so will need to be re-processed to achieve this.


bcbio-nextgen automates the calling and validation used in this comparison. We aim to make it easy to install, use and extend.

For GATK HaplotypeCaller based joint genotyping, we implement the GATK best practices recommended by the Broad. Individual sample variant calls produce a gVCF output file that contains both variants as well as probability information about reference regions. Next, variants are jointly called using GenotypeGVFs to produce the final population VCF file.

For the other supported callers – FreeBayes, Platypus and samtools – we use a generalized recalling approach, implemented in bcbio.variation.recall. bcbio-nextgen first calls each individual sample as a standard VCF. We then combine these individual sample VCFs into a global summary of all variant positions called across all samples. Finally we recall at each potential variant position, producing a globally squared off joint callset for the sample that we merge into the final joint VCF. This process parallelizes by chromosome region and by sample, allowing efficient use of resources in both clusters and large multiple core machines.

bcbio.variation.recall generalizes to any variant caller that supports recalling with an input set of variants. Knowing the context of potential variants helps inform better calling. This method requires having the individual sample BAM file available to perform recalling. Having the reads present does provide the ability to improve recalling by taking advantage of realigning reads into haplotypes given known variants, an approach we’ll explore more in future work. The implementation is also general and could support gVCF based combining as this becomes available for non-GATK callers.

Generalized joint calling

We evaluated all callers against the NA12878 Genome in a Bottle reference standard using the NA12878/NA12891/NA12892 trio from the CEPH 1463 Pedigree, with 50x whole genome coverage from Illumina’s platinum genomes. The validation provides putative true positives (concordant), false negatives (discordant missing), and false positives (discordant extra) for all callers:

Incremental joint calling: GATK HaplotypeCaller, FreeBayes, Platypus, samtools

Overall, there is not a large difference in sensitivity and precision for the four methods, giving us four high-quality options for performing joint variant calling on germline samples. The post-calling filters provide similar levels of false positives to enable comparisons of sensitivity. Notably, samtools new calling method is now as good as other approaches, in contrast with previous evaluations, demonstrating the value of continuing to improve open source tools and having updated benchmarks to reflect these improvements.

Improving sensitivity and precision is always an ongoing process and this evaluation identifies some areas to focus on for future work:

  • Platypus SNP and indel calling is slightly less sensitive than other approaches. We worked on Platypus calling parameters and post-call filtering to increase sensitivity from the defaults without introducing a large number of false positives, but welcome suggestions for more improvements.
  • samtools indel calling needs additional work to reduce false positive indels in joint and pooled calling. There is more detail on this below in the comparison with single sample samtools calling.

Joint versus pooled versus single approaches

We validated the same NA12878 trio with pooled and single sample calling to assess the advantages of joint calling over single sample, and whether joint calling is comparable in quality to calling simultaneously. The full evaluation for pooled calling shows that performance is similar to joint methods:

Pooled calling: GATK HaplotypeCaller, FreeBayes, Platypus, samtools

If you plot joint, pooled and single sample calling next to each other there are some interesting small differences between approaches that identify areas for further improvement. As an example, here are GATK HaplotypeCaller and samtools with the three approaches presented side by side:

Joint, pooled and single calling: GATK HaplotypeCaller and samtools

GATK HaplotypeCaller sensitivity and precision are close between the three methods, with small trade offs for different methods. For SNPs, pooled calling is most sensitive at the cost of more false positives, and single calling is more precise at the cost of some sensitivity. Joint calling is intermediate between these two extremes. For indels, joint calling is the most sensitive at the cost of more false positives, with pooled calling falling between joint and single sample calling.

For samtools, precision is currently best tuned for single sample calling. Pooled calling provides better sensitivity, but at the cost of a larger number of false positives. The joint calling implementation regains a bit of this sensitivity but still suffers from increased false positives. The authors of samtools tuned variant calling nicely for single samples, but there are opportunities to increase sensitivity when incorporating multiple samples via a joint method.

Generally, we don’t expect the same advantages for pooled or joint calling in a trio as we’d see in a larger population. However, even for this small evaluation population we can see the improvements available by considering additional variant information from other samples. For Platypus we unexpectedly had better calls from joint calling compared to pooled calling, but expect these differences to harmonize over time as the tools continue to improve.

Overall, this comparison identifies areas where we can hope to improve generalized joint calling. We plan to provide specific suggestions and feedback to samtools, Platypus and other tool authors as part of a continuous validation and feedback process.

Reproducing and extending the analysis

All variant callers and calling methods validated here are available for running in bcbio-nextgen. bcbio automatically installs the generalized joint calling implementation, and it is also available as a java executable at bcbio.variation.recall. All tools are freely available, open source and community developed and we welcome your feedback and contributions.

The documentation contains full instructions for running the joint analysis. This is an extended version of previous work on validation of trio calling and uses the same input dataset with a bcbio configuration that includes single, pooled and joint calling:

mkdir -p NA12878-trio-eval/config NA12878-trio-eval/input NA12878-trio-eval/work-joint
cd NA12878-trio-eval/config
cd ../input
cd ../work_joint ../config/NA12878-trio-wgs-joint.yaml -n 16

Having a general joint calling implementation with good sensitivity and precision is a starting point for more research and development. To build off this work we plan to:

  • Provide better ensemble calling methods that scale to large multi-sample calling projects.
  • Work with FreeBayes, Platypus and samtools tool authors to provide support for gVCF style files to avoid the need to have BAM files present during joint calling, and to improve sensitivity and precision during recalling-based joint approaches.
  • Combine variant calls with local reassembly to improve sensitivity and precision. Erik Garrison’s glia provides streaming local realignment given a set of potential variants. Jared Simpson used the SGA assembler to combine FreeBayes calls with de-novo assembly. Ideally we could identify difficult regions of the genome based on alignment information and focus more computationally expensive assembly approaches there.

We plan to continue working with the open source scientific community to integrate, extend and improve these tools and are happy for any feedback and suggestions.

Written by Brad Chapman

October 7, 2014 at 8:53 am

Validated whole genome structural variation detection using multiple callers

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Structural variant detection goals

This post describes community based work integrating structural variant calling and validation into bcbio-nextgen. I’ve previously written about approaches for validating single nucleotide changes (SNPs) and small insertions/deletions (Indels), but it has always been unfortunate to not have reliable ways to detect larger structural variations: deletions, duplications, inversions, translocations and other disruptive events. Detecting these events with short read sequencing is difficult, and our goal in bcbio is to create a global summary of predicted structural variants from multiple callers alongside measures of sensitivity and precision.

The latest release of bcbio automates structural variant calling with three callers:

bcbio integrates structural variation predictions from all approaches into a high level BED file. This is a first pass way to identify potentially disruptive large scale events. Here are example regions: a duplication called by all 3 callers, a deletion called by 2 callers, and a complex region with both deletions and duplications.

9  139526855 139527537 DUP_delly,DUP_lumpy,cnv3_cn_mops
10  99034861  99037400 DEL_delly,cnv0_cn_mops,cnv1_cn_mops
12   8575814   8596742 BND_lumpy,DEL_delly,DEL_lumpy,DUP_delly,DUP_lumpy,cnv1_cn_mops,cnv3_cn_mops

This output associates larger structural events with regions of interest in a high level way, while allowing us to quickly determine the individual tool support for each event. Using this, we are no longer blind to potential structural changes and can use the summary to target in-depth investigation with the more detailed metrics available from each caller and a read viewer like PyBamView. The results can also help inform prioritization of SNP and Indel calls since structural rearrangements often associate with false positives. Longer term we hope this structural variant summary and comparison work will be useful for community validation efforts like the Global Alliance for Genomics and Health benchmarking group, the Genome in a Bottle consortium and the ICGC-TCGA DREAM Mutation Calling challenge.

Below I’ll describe a full evaluation of the sensitivity and precision of this combined approach using an NA12878 trio, as well as describe how to run and extend this work using bcbio-nextgen.

This blog post is the culmination of a lot of work and support from the open source bioinformatics community. David Jenkins worked with our our group for the summer and headed up evaluation of structural variation results. We received wonderful support from Colby Chang, Ryan Layer, Ira Hall and Aaron Quinlan on both LUMPY and structural variation validation in general. They freely shared scripts and datasets for evaluation, which gave us the materials to make these comparisons. Günter Klambauer gave us great practical advice on using cn.mops. Tobias Rausch helped immensely with tips for speeding up DELLY on whole genomes, and Ashok Ragavendran from Mike Talkowski’s lab generously discussed tricks for scaling DELLY runs. Harvard Research Computing provided critical support and compute for this analysis as part of a collaboration with Intel.


To validate the output of this combined structural variant calling approach we used a set of over 4000 validated deletions made available by Ryan Layer as part of the LUMPY manuscript. These are deletion calls in NA12878 with secondary support evidence from Moleculo and PacBio datasets. We further subset these regions by removing calls in low complexity regions identified in Heng Li’s variant calling artifacts paper. An alternative set of problematic regions are the potential misassembly regions identified by Colby Chang and Ira Hall during LUMPY and speedseq development (Edit: The original post mistakenly mentioned these overlap significantly with low complexity regions, but that is only due to overlap in obvious problem areas like the N gap regions. We’ll need additional work to incorporate both regions into future evaluations. Thanks to Colby for catching this error.). These are a useful proxy for regions we’re currently not able to reliably call structural variants in.

We ran all structural variant callers using the NA12878/NA12891/NA12892 trio from the CEPH 1463 Pedigree as an input dataset. This consists of 50x whole genome reads from Illumina’s platinum genomes project, and is the same dataset used in our previous analyses of population based SNP and small indel calling.

Our goal is to define boundaries on sensitivity – the percentage of validated calls we detect – and precision – how many of the total calls overlap with validated regions. We required a simple overlap of the called regions with validated regions to consider a called variant as validated, and stratified results by event size to quantify detection metrics at different size ranges.

The comparison highlights the value of providing a combined call set. I’d caution against using this as a comparison between methods. Accurate structural variation calling depends on multiple sources of evidence and we still have work to do in improving our ability to filter for maximal sensitivity and specificity. The ensemble method in the figure below displays results of our final calls, made from collapsing structural variant calls from all three input callers:

Structural variant calling sensitivity and precision at different event sizes

Across all size classes, we detect approximately half of the structural variants and expect that about half of the called events are false positives. Smaller structural variants of less than 1kb are the most difficult to detect with these methods. Larger events from 1kb to 25kb have better sensitivity and precision. As the size of the events increase precision decreases, so larger called events tend to have more false positives.

Beyond the values for sensitivity and precision, the biggest takeaway is that combining multiple callers helps detect additional variants we’d miss with any individual caller. Count based callers like cn.mops enable improved sensitivity on large deletions but don’t resolve small deletions at 50x depth using our current settings, although tuning can help detect these smaller sized events as well. Similarly, lumpy and delly capture different sets of variants across all of the size classes.

The comparison also emphasizes the potential for improving both individual caller filtering and ensemble structural variant preparation. The ensemble method uses bedtools to create a merged superset of all individually called regions. This is the simplest possible approach to combine calls. Similarly, individual caller filters are intentionally simple as well. cn.mops calling performs no additional filtering beyond the defaults, and could use adjustment to detect and filter smaller events. Our DELLY filter requires 4 supporting reads or both split and paired read evidence. Our LUMPY filter require at least 4 supporting reads to retain an event. We welcome discussion of the costs and tradeoffs of these approaches. For instance, requiring split and paired evidence for DELLY increases precision at the cost of sensitivity. These filters are a useful starting point and resolution, but we hope to continue to refine and improve them over time.


bcbio-nextgen handles installation and automation of the programs used in this comparison. The documentation contains instructions to download the data and run the NA12878 trio calling and validation. This input configuration file should be easily adjusted to run on your data of interest.

The current implementation has reasonable run times for whole genome structural variant calling. We use samblaster to perform duplicate marking alongside identification of discordant and split read pairs. The aligned reads from bwa stream directly into samblaster, adding minimal processing time to the run. For LUMPY calling, the pre-prepared split and discordant reads feed directly into speedseq, which nicely automates the process of running LUMPY. For DELLY, we subsample correct pairs in the input BAM to 50 million reads and combine with the pre-extracted problematic pairs to improve runtimes for whole genome inputs.

We processed three concurrently called 50x whole genome samples from FASTQ reads to validated structural variants in approximately 3 days using 32 cores. Following the preparation work described above, LUMPY calling took 6 hours, DELLY takes 24 hours parallelized on 32 cores and cn.mops took 16 hours parallelized by chromosome on 16 cores. This is a single data point for current capabilities, and is an area where we hope to continue to improve scalability and parallelization.

The implementation and validation are fully integrated into the community developed bcbio-nextgen project and we hope to expand this work to incorporate additional structural variant callers like Pindel and CNVkit, as well as improving filtering and ensemble calling. We also want to expand structural variant validation to include tumor/normal cancer samples and targeted sequencing. We welcome contributions and suggestions on current and future directions in structural variant calling.

Written by Brad Chapman

August 12, 2014 at 1:22 pm

Whole genome trio variant calling evaluation: low complexity regions, GATK VQSR and high depth filters

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Whole genome trio validation

I’ve written previously about the approaches we use to validate the bcbio-nextgen variant calling framework, specifically evaluating aligners and variant calling methods and assessing the impact of BAM post-alignment preparation methods. We’re continually looking to improve both the pipeline and validation methods and two recent papers helped advance best-practices for evaluating and filtering variant calls:

  • Michael Linderman and colleagues describe approaches for validating clinical exome and whole genome sequencing results. One key result I took from the paper was the difference in assessment between exome and whole genome callsets. Coverage differences due to capture characterize discordant exome variants, while complex genome regions drive whole genome discordants. Reading this paper pushed us to evaluate whole genome population based variant calling, which is now feasible due to improvements in bcbio-nextgen scalability.
  • Heng Li identified variant calling artifacts and proposed filtering approaches to remove them, as well as characterizing caller error rates. We’ll investigate two of the filters he proposed: removing variants in low complexity regions, and filtering high depth variants.

We use the NA12878/NA12891/NA12892 trio from the CEPH 1463 Pedigree as an input dataset, consisting of 50x whole genome reads from Illumina’s platinum genomes. This enables both whole genome comparisons, as well as pooled family calling that replicates best-practice for calling within populations. We aligned reads using bwa-mem and performed streaming de-duplication detection with samblaster. Combined with no recalibration or realignment based on our previous assessment, this enabled fully streamed preparation of BAM files from input fastq reads. We called variants using two realigning callers: FreeBayes (v0.9.14-7) and GATK HaplotypeCaller (3.1-1-g07a4bf8) and evaluated calls using the Genome in a Bottle reference callset for NA12878 (v0.2-NIST-RTG). The bcbio-nextgen documentation has full instructions for reproducing the analysis.

This work provides three practical improvements for variant calling and validation:

  • Low complexity regions contribute 45% of the indels in whole genome evaluations, and are highly variable between callers. This replicates Heng’s results and Michael’s assessment of common errors in whole genome samples, and indicates we need to specifically identify and assess the 2% of the genome labeled as low complexity. Practically, we’ll exclude them from further evaluations to avoid non-representative bias, and suggest removing or flagging them when producing whole genome variant calls.
  • We add a filter for removing false positives from FreeBayes calls in high depth, low quality regions. This removes variants in high depth regions that are likely due to copy number or other larger structural events, and replicates Heng’s filtering results.
  • We improved settings for GATK variant quality recalibration (VQSR). The default VQSR settings are conservative for SNPs and need adjustment to be compatible with the sensitivity available through FreeBayes or GATK using hard filters.

Low complexity regions

Low complexity regions (LCRs) consist of locally repetitive sections of the genome. Heng’s paper identified these using mdust and provides a BED file of LCRs covering 2% of the genome. Repeats in these regions can lead to artifacts in sequencing and variant calling. Heng’s paper provides examples of areas where a full de-novo assembly correctly resolves the underlying structure, while local reassembly variant callers do not.

To assess the impact of low complexity regions on variant calling, we compared calls from FreeBayes and GATK HaplotypeCaller to the Genome in a Bottle reference callset with and without low complexity regions included. The graph below shows concordant non-reference variant calls alongside discordant calls in three categories: missing discordants are potential false negatives, extras are potential false positives, and shared are variants that overlap between the evaluation and reference callsets but differ due to zygosity (heterozygote versus homozygote) or indel representation.

Low complexity regions for GATK and FreeBayes validation

  • For SNPs, removing low complexity regions removes approximately ~2% of the total calls for both FreeBayes and GATK. This corresponds to the 2% of the genome subtracted by removing LCRs.
  • For indels, removing LCRs removes 45% of the calls due to the over-representation of indels in repeat regions. Additionally, this results in approximately equal GATK and FreeBayes concordant indels after LCR removal. Since the Genome in a Bottle reference callset uses GATK HaplotypeCaller to resolve discrepant calls, this change in concordance is likely due to bias towards GATK’s approaches for indel resolution in complex regions.
  • The default GATK VQSR calls for SNPs are not as sensitive, relative to FreeBayes calls. I’ll describe additional work to improve this below.

Practically, we’ll now exclude low complexity regions in variant comparisons to avoid potential bias and more accurately represent calls in the remaining non-LCR genome. We’ll additionally flag low complexity indels in non-evaluation callsets as likely to require additional followup. Longer term, we need to incorporate callers specifically designed for repeats like lobSTR to more accurately characterize these regions.

High depth, low quality, filter for FreeBayes

The second filter proposed in Heng’s paper was removal of high depth variants. This was a useful change in mindset for me as I’ve primarily thought about removing low quality, low depth variants. However, high depth regions can indicate potential copy number variations or hidden duplicates which result in spurious calls.

Comparing true and false positive FreeBayes calls with a pooled multi-sample call quality of less than 500 identifies a large grouping of false positive heterozygous variants at a combined depth, across the trio, of 200:

Heterozygotes by depth and quality: true versus false positive

The cutoff proposed by Heng was to calculate the average depth of called variants and set the cutoff as the average depth plus a multiplier of 3 or 4 times the square root of average depth. This dataset was an average depth of 169 for the trio, corresponding to a cutoff of 208 if we use the 3 multiplier, which compares nicely with a manual cutoff you’d set looking at the above graphs. Applying a cutoff of QUAL < 500 and DP > 208 produces a reduction in false positives with little impact on sensitivity:

Improvement in filtering false positives with high depth filter

A nice bonus of this filter is that it makes intuitive sense: variants with high depth and low quality indicate there is something problematic, and depth manages to partially compensate for the underlying issue. Inspired by GATK’s QualByDepth annotation and default filter of QD < 2.0, we incorporated a generalized version of this into bcbio-nextgen’s FreeBayes filter: QUAL < (depth-cutoff * 2.0) and DP > depth-cutoff.

GATK variant quality score recalibration (VQSR)

The other area where we needed to improve was using GATK Variant Quality Score Recalibration. The default parameters provide a set of calls that are overly conservative relative to the FreeBayes calls. VQSR provides the ability to tune the filtering so we experimented with multiple configurations to achieve approximately equal sensitivity relative to FreeBayes for both SNPs and Indels. The comparisons use the Genome in a Bottle reference callset for evaluation, and include VQSR default settings, multiple tranche levels and GATK’s suggested hard filters:

VQSR tuning: SNPs VQSR tuning: indels

While the sensitivity/specificity tradeoff depends on the research question, in trying to set a generally useful default we’d like to be less conservative than the GATK VQSR default. We learned these tips and tricks for tuning VQSR filtering:

  • The default setting for VQSR is not a tranche level (like 99.0), but rather a LOD score of 0. In this experiment, that corresponded to a tranche of ~99.0 for SNPs and ~98.0 for indels. The best-practice example documentation uses command line parameter that specify a consistent tranche of 99.0 for both SNPs and indels, so depending on which you follow as a default you’ll get different sensitivities.
  • To increase sensitivity, increase the tranche level. My expectations were that decreasing the tranche level would include more variants, but that actually applies additional filters. My suggestion for understanding tranche levels is that they specify the percentage of variants you want to capture; a tranche of 99.9% captures 99.9% of the true cases in the training set, while 99.0% captures less.
  • We found tranche settings of 99.97% for SNPs and 98.0% for indels correspond to roughly the sensitivity/specificity that you achieve with FreeBayes. These are the new default settings in bcbio-nextgen.
  • Using hard filtering of variants based on GATK recommendations performs well and is also a good default choice. For SNPs, the hard filter defaults are less conservative and more in line with FreeBayes results than VQSR defaults. VQSR has improved specificity at the same sensitivity and has the advantage of being configurable, but will require an extra tuning step.

Overall VQSR provides good filtering and the ability to tune sensitivity but requires validation work to select tranche cutoffs that are as sensitive as hard filter defaults, since default values tend to be overly conservative for SNP calling. In the absence of the ability or desire to tune VQSR tranche levels, the GATK hard filters provide a nice default without much of a loss in precision.

Data availability and future work

Thanks to continued community work on improving variant calling evaluations, this post demonstrates practical improvements in bcbio-nextgen variant calling. We welcome interested contributors to re-run and expand on the analysis, with full instructions in the bcbio-nextgen example pipeline documentation. Some of the output files from the analysis may also be useful:

  • VCF files for FreeBayes true positive and false positive heterozygote calls, used here to improve filtering via assessment of high depth regions. Heterozygotes make up the majority of false positive calls so take the most work to correctly filter and detect.
  • Shared false positives from FreeBayes and GATK HaplotypeCaller. These are potential missing variants in the Genome in a Bottle reference. Alternatively, they may represent persistent errors found in multiple callers.

We plan to continue to explore variant calling improvements in bcbio-nextgen. Our next steps are to use the trio population framework to compared pooled population calling versus the incremental joint discovery approach introduced in GATK 3. We’d also like to compare with single sample calling followed by subsequent squaring off/backfilling to assess the value of concurrent population calling. We welcome suggestions and thoughts on this work and future directions.

Written by Brad Chapman

May 12, 2014 at 6:03 am

Updated comparison of variant detection methods: Ensemble, FreeBayes and minimal BAM preparation pipelines

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Variant evaluation overview

I previously discussed our approach for evaluating variant detection methods using a highly confident set of reference calls provided by NIST’s Genome in a Bottle consortium for the NA12878 human HapMap genome, In this post, I’ll update those conclusions based on recent improvements in GATK and FreeBayes.

The comparisons use bcbio-nextgen, an automated open-source pipeline for variant calling and evaluation that identifies concordant and discordant variants with the XPrize validation protocol. By having an automated validation workflow attached to a regularly updated, community developed, variant calling pipeline, we can actively track progress of variant callers and provide updates as algorithms improve.

Since the initial post, There have been two new GATK releases of UnifiedGenotyper and HaplotypeCaller, as well as multiple improvements to FreeBayes. Additionally we’ve enchanced our ensemble calling method, which combines inputs from multiple callers into a single final set of calls, to better handle comparisons with inputs from three callers.

The goal of this post is to re-evaluate these variant detection approaches and provide an updated set of recommendations:

  • FreeBayes detects more concordant SNPs and indels compared to GATK approaches, including GATK’s HaplotypeCaller method.
  • Post-alignment BAM processing steps like base quality recalibration and realignment have little impact on the quality of variant calls with variant callers that perform local realignment, including FreeBayes and GATK HaplotypeCaller.
  • The Ensemble calling method provides the best variant detection by combining inputs from GATK UnifiedGenotyper, HaplotypeCaller and FreeBayes.

Avoiding the post-alignment BAM recalibration and realignment steps allows us to save significant time and pipeline complexity. Combined with the improvements in FreeBayes, this enables a variant calling pipeline that can be freely used for academic, clinical and commercial work with equal quality variant calls compared to current GATK best-practice approaches.

Calling and evaluation methods

We called variants on a NA12878 exome dataset from EdgeBio’s clinical pipeline and assessed them against the NIST’s Genome in a Bottle reference material. Full instructions for replicating the analysis and installing the pipeline are available from the bcbio-nextgen documentation site. Following alignment with bwa-mem (0.7.5a), we post-processed the BAM files with two methods:

We then called variants with three general purpose callers:

Finally, we evaluated the calls from each combination of variant caller and BAM post-alignment preparation method using the bcbio.variation framework. This provides a summary identifying concordant and discordant variants, separating SNPs and indels since they have different error profiles. Additionally it classifies discordant variants. where the reference material and evaluation variants differ, into three categories:

  • Extra variants, called in the evaluation data but not in the reference. These are potential false positives or missing calls from the reference materials.
  • Missing variants, found in the NA12878 reference but not in the evaluation data set. These are potential false negatives.
  • Shared variants, called in both the evaluation and reference but differently represented. This results from allele differences, such as heterozygote versus homozygote calls, or variant identification differences, such as indel start and end coordinates.

Variant caller comparison

Using this framework, we compared the 3 variant callers and combined ensemble method:

  • FreeBayes outperforms the GATK callers on both SNP and indel calling. The most recent versions of FreeBayes have improved sensitivity and specificity which puts them on par with GATK HaplotypeCaller. One area where FreeBayes performs better is in correctly resolving heterozygote/homozygote calls, reflected in the lower number of discordant shared variants.
  • GATK HaplotypeCaller is all around better than the UnifiedGenotyper. In the previous comparison, we found UnifiedGenotyper performed better on SNPs and HaplotypeCaller better on indels, but the recent improvements in GATK 2.7 have resolved the difference in SNP calling. If using a GATK pipeline, UnifiedGenotyper lags behind the realigning callers in resolving indels, and I’d recommend using HaplotypeCaller. This mirrors the GATK team’s current recommendations.
  • The ensemble calling approach provides the best overall resolution of both SNPs and indels. The one area where it lags slightly behind is in identification of homozygote/heterozygote calls, especially in indels. This is due to positions where HaplotypeCaller and FreeBayes both call variants but differ on whether it is a heterozygote or homozygote, reflected as higher discordant shared counts.

Comparison of variant callers, GATK best practice preparation

In addition to calling sensitivity and specificity, an additional factor to consider is the required processing time. Rough benchmarks on family-based calling of whole genome sequencing data indicate that HaplotypeCaller is roughly 7x slower than UnifiedGenotyper and FreeBayes is 2x slower. On multiple 30x whole genome samples, our experience is that calling can range from 10 hours for GATK UnifiedGenotyper to 70 hours for HaplotypeCallers. Ensemble calling requires running all three callers plus combining into a final call set, and for family-based whole genome samples can add another 100 hours of processing time. These estimates fluctuate greatly depending on the compute infrastructure and presence of longer difficult genomic regions with deeper coverage, but give some estimates of timing considerations.

Post-alignment BAM preparation comparison

Given the improved accuracy of local realignment haplotype-based callers like FreeBayes and HaplotypeCaller, we explored the accuracy cost of removing the post-alignment BAM processing steps. The recommended GATK best-practice is to follow up alignment with identification of duplicate reads, followed by base quality score recalibration and realignment around indels. Based on whole genome benchmarking work, these steps can take as long as the initial alignment and scale poorly due to the high IO costs of manipulating large BAM files. For multiple 30x whole genome samples running on 16 cores per sample, this can account for 12 to 16 hours of processing time.

To compare the quality impact of avoiding recalibration and realignment, we performed the identical alignment and variant calling steps as above, but did minimal post-alignment BAM preparation. Following alignment, the only step performed was deduplication using samtools rmdup. Unlike Picard MarkDuplicates, samtools rmdup handles piped streaming input to avoid IO penalties. This is at the cost of not handling some edge cases. Longer term, we’d like to explore biobambam’s markduplicates2, which implements a more efficient streaming version of the Picard MarkDuplicates algorithm.

Suprisingly, skipping base recalibration and indel realignment had almost no impact on the quality of resulting variant calls:

Comparison of variant callers, minimal post-alignment preparation

While GATK UnifiedGenotyper suffers during indel calling without recalibration and realignment, both HaplotypeCaller and FreeBayes perform as good or better without these steps. This allows us to save on processing time and complexity without sacrificing call quality when using a haplotype aware realigning caller.

Caveats and conclusions

Taken together, the improvements in FreeBayes and ability to avoid post-alignment BAM processing allow use of a commercially unrestricted GATK-free pipeline with equal quality to current GATK best practices. Adding in GATK’s two callers plus our ensemble combining method provides the most accurate overall calls, at the cost of additional processing time.

It’s also important to consider potential drawbacks of this analysis as we continue to design future evaluations. The comparison is in exome regions for single sample variant calling. In future work it would be helpful to have population or family based inputs. We’d also like to prepare test datasets that focus specifically on evaluating the quality of calls in more difficult repetitive regions within the whole genome. Using populations or whole genomes would also allow use of GATK’s Variant Quality Score Recalibration as part of the pipeline, which could provide improved filtering compared to the hard-filtering approach used here.

Another consideration is that the reference callset prepared by the Genome in a Bottle consortium makes extensive use of GATK tools during preparation. Evaluation of the reference materials with FreeBayes and other callers can help reduce potential GATK-specific biases when continuing to develop reliable reference materials.

All of these pipelines are freely available, open-source, community developed projects and we welcome feedback and contributors. By integrating validation into a scalable analysis pipeline, we hope to build a community interested in widely accessible calling pipelines coupled with well-evaluated reference datasets and methods.

Written by Brad Chapman

October 21, 2013 at 6:35 am

Posted in variation

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Scaling variant detection pipelines for whole genome sequencing analysis

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Scaling for whole genome sequencing

Moving from exome to whole genome sequencing introduces a myriad of scaling and informatics challenges. In addition to the biological component of correctly identifying biological variation, it’s equally important to be able to handle the informatics complexities that come with scaling up to whole genomes.

At Harvard School of Public Health, we are processing an increasing number of whole genome samples and the goal of this post is to share experiences scaling the bcbio-nextgen pipeline to handle the associated increase in file sizes and computational requirements. We’ll provide an overview of the pipeline architecture in bcbio-nextgen and detail the four areas we found most useful to overcome processing bottlenecks:

  • Support heterogeneous cluster creation to maximize resource usage.
  • Increase parallelism by developing flexible methods to split and process by genomic regions.
  • Avoid file IO and prefer streaming piped processing pipelines.
  • Explore distributed file systems to better handle file IO.

This overview isn’t meant as a prescription, but rather as a description of experiences so far. The work is a collaboration between the HSPH Bioinformatics Core, the research computing team at Harvard FAS and Dell Research. We welcome suggestions and thoughts from others working on these problems.

Pipeline architecture

The bcbio-nextgen pipeline runs in parallel on single multicore machines or distributed on job scheduler managed clusters like LSF, SGE, and TORQUE. The IPython parallel framework manages the set up of parallel engines and handling communication between them. These abstractions allow the same pipeline to scale from a single processor to hundreds of node on a cluster.

The high level diagram of the analysis pipeline shows the major steps in the process. For whole genome samples we start with large 100Gb+ files of reads in FASTQ or BAM format and perform alignment, post-alignment processing, variant calling and variant post processing. These steps involve numerous externally developed software tools with different processing and memory requirements.

Variant calling overview

Heterogeneous clusters

A major change in the pipeline was supporting creation of heterogeneous processing environments targeted for specific programs. This moves away from our previous architecture, which attempted to flatten processing and utilize single cores throughout. Due to algorithm restrictions, some software requires the entire set of reads for analysis. For instance, GATK’s base quality recalibrator uses the entire set of aligned reads to accurately calculate inputs for read recalibration. Other software operates more efficiently on entire files: the alignment step scales better by running using multiple cores on a single machine, since the IO penalty for splitting the input file is so severe.

To support this, bcbio-nextgen creates an appropriate type of cluster environment for each step:

  • Multicore: Allocates groups of same machine processors, allowing analysis of individual samples with multiple cores. For example, this enables running bwa alignment with 16 cores on multiprocessor machines.
  • Full usage of single cores: Maximize usage of single cores for processes that scale beyond the number of samples. For example, we run variant calling in parallel across subsets of the genome.
  • Per sample single core usage: Some steps do not currently parallelize beyond the number of samples, so require a single core per sample.

IPython parallel provides the distributed framework for creating these processing setups, working on top of existing schedulers like LSF, SGE and TORQUE. It creates processing engines on distributed cores within the cluster, using ZeroMQ to communicate job information between machines.

Cluster schedulers allow this type of control over core usage, but an additional future step is to include memory and disk IO requirements as part of heterogeneous environment creation. Amazon Web Services allows selection of exact memory, disk and compute resources to match the computational step. Eucalyptus and OpenStack bring this control to local hardware and virtual machines.

Variant calling overview

Parallelism by genomic regions

While the initial alignment and preparation steps require analysis of a full set of reads due to IO and algorithm restrictions, subsequent steps can run with increased parallelism by splitting across genomic regions. Variant detection algorithms do require processing continuous blocks of reads together, allowing local realignment algorithms to correctly characterize closely spaced SNPs and indels. Previously, we’d split analyses by chromosome but this has the downside of tying analysis times to chromosome 1, the largest chromosome.

The pipeline now identifies chromosome blocks without callable reads. These blocks group by either genomic features like repetitive hard to align sequence or analysis requirements like defined target regions. Using the globally shared callable regions across samples, we fraction the genome into more uniform sections for processing. As a result we can work on smaller chunks of reads during time critical parts of the process: applying base recalibration, de-duplication, realignment and variant calling.

Parallel block selection from genome

Streaming pipelines

A key bottleneck throughout the pipeline is disk usage. Steps requiring reading and writing large BAM or FASTQ files slow down dramatically once they overburden disk IO, distributed filesystem capabilities or ethernet connectivity between storage nodes. A practical solution to this problem is to avoid intermediate files and use unix pipes to stream results between processes.

We reworked our alignment step specifically to eliminate these issues. The previous attempt took a disk centric approach that allowed scaling out to multiple single cores in a cluster. We split an input FASTQ or BAM file into individual chunks of reads, and then aligned each of these chunks independently. Finally, we merged all the individual BAMs together to produce a final BAM file to pass on to the next step in the process. While nicely generalized, it did not scale when running multiple concurrent whole genomes.

The updated pipeline uses multicore support in samtools and aligners like bwa-mem and novoalign to pipe all steps as a stream: preparation of input reads, alignment, conversion to BAM and coordinate sorting of aligned reads. This results in improved scaling at the cost of only being able to increase single sample throughput to the maximum processors on a machine.

More generally, the entire process creates numerous temporary file intermediates that are a cause of scaling issues. Commonly used best-practice toolkits like Picard and GATK primarily require intermediate files. In contrast, tools in the Marth lab’s gkno pipeline handle streaming input and output making it possible to create alignment post-processing pipelines which minimize temporary file creation. As a general rule, supporting streaming algorithms amenable to piping can ameliorate file load issues associated with scaling up variant calling pipelines. This echos the focus on streaming algorithms Titus Brown advocates for dealing with large metagenomic datasets.

Distributed file systems

While all three of CPU, memory and disk speed limit individual steps during processing, the hardest variable to tweak is disk throughput. CPU and memory limitations have understandable solutions: buy faster CPUs and more memory. Improving disk access is not as easily solved, even with monetary resources, as it’s not clear what combination of disk and distributed filesystem will produce the best results for this type of pipeline.

We’ve experimented with NFS, GlusterFS and Lustre for handling disk access associated with high throughput variant calling. Each requires extensive tweaking and none has been unanimously better for all parts of the process. Much credit is due to John Morrissey and the research computing team at Harvard FAS for help performing incredible GlusterFS and network improvements as we worked through scaling issues, and Glen Otero, Will Cottay and Neil Klosterman at Dell for configuring an environment for NFS and Lustre testing. We can summarize what we’ve learned so far in two points:

  • A key variable is the network connectivity between storage nodes. We’ve worked with the pipeline on networks ranging from 1 GigE to InfiniBand connectivity, and increased throughput delays scaling slowdowns.
  • Different part of the processes stress different distributed file systems in complex ways. NFS provides the best speed compared to single machine processing until you hit scaling issues, then it slows down dramatically. Lustre and GlusterFS result in a reasonable performance hit for less disk intensive processing, but delay the dramatic slowdowns seen with NFS. However, when these systems reach their limits they hit a slowdown wall as bad or worse than NFS. One especially slow process identified on Gluster is SQLite indexing, although we need to do more investigation to identify specific underlying causes of the slowdown.

Other approaches we’re considering include utilizing high speed local temporary disk, reducing writes to long term distributed storage file systems. This introduces another set of challenges: avoiding stressing or filling up local disk when running multiple processes. We’ve also had good reports about using MooseFS but haven’t yet explored setting up and configuring another distributed file system. I’d love to hear experiences and suggestions from anyone with good or bad experiences using distributed file systems for this type of disk intensive high throughput sequencing analysis.

A final challenge associated with improving disk throughput is designing a pipeline that is not overly engineered to a specific system. We’d like to be able to take advantage of systems with large SSD attached temporary disk or wonderfully configured distributed file systems, while maintaining the ability scale on other systems. This is critical for building a community framework that multiple groups can use and contribute to.

Timing results

Providing detailed timing estimates for large, heterogeneous pipelines is difficult since they will be highly depending on the architecture and input files. Here we’ll present some concrete numbers that provide more insight into the conclusions presented above. These are more useful as a side by side comparison between approaches, rather than hard numbers to predict scaling on your own systems.

In partnership with Dell Solutions Center, we’ve been performing benchmarking of the pipeline on dedicated cluster hardware. The Dell system has 32 16-core machines connected with high speed InfiniBand to distributed NFS and Lustre file systems. We’re incredibly appreciative of Dell’s generosity in configuring, benchmarking and scaling out this system.

As a benchmark, we use 10x coverage whole genome human sequencing data from the Illumina platinum genomes project. Detailed instructions on setting up and running the analysis are available as part of the bcbio-nextgen example pipeline documentation.

Below are wall-clock timing results, in total hours, for scaling from 1 to 30 samples on both Lustre and NFS fileystems:

primary 1 sample 1 sample 1 sample 30 samples 30 samples
bottle 16 cores 96 cores 96 cores 480 cores 480 cores
neck Lustre Lustre NFS Lustre NFS
alignment cpu/mem 4.3h 4.3h 3.9h 4.5h 6.1h
align post-process io 3.7h 1.0h 0.9h 7.0h 20.7h
variant calling cpu/mem 2.9h 0.5h 0.5h 3.0h 1.8h
variant post-process io 1.0h 1.0h 0.6h 4.0h 1.5h
total 11.9h 6.8h 5.9h 18.5h 30.1h

Some interesting conclusions:

  • Scaling single samples to additional cores (16 to 96) provides a 40% reduction in processing time due to increased parallelism during post-processing and variant calling.
  • Lustre provides the best scale out from 1 to 30 samples, with 30 sample concurrent processing taking only 1.5x as along as a single sample.
  • NFS provides slightly better performance than Lustre for single sample scaling.
  • In contrast, NFS runs into scaling issues at 30 samples, proceeding 5.5 times slower during the IO intensive alignment post-processing step.

This is preliminary work as we continue to optimize code parallelism and work on cluster and distributed file system setup. We welcome feedback and thoughts to improve pipeline throughput and scaling recommendations.

Written by Brad Chapman

May 22, 2013 at 6:50 am

Posted in variation

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Framework for evaluating variant detection methods: comparison of aligners and callers

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Variant detection and grading overview

Developing pipelines for detecting variants from high throughput sequencing data is challenging due to rapidly changing algorithms and relatively low concordance between methods. This post will discuss automated methods providing evaluation of variant calls, enabling detailed diagnosis of discordant differences between multiple calling approaches. This allows us to:

  • Characterize strengths and weaknesses of alignment, post-alignment preparation and calling methods.
  • Automatically verify pipeline updates and installations to ensure variant calls recover expected variations. This extends the XPrize validation protocol to provide full summary metrics on concordance and discordance of variants.
  • Make recommendations on best-practice approaches to use in sequencing studies requiring either exome or whole genome variant calling.
  • Identify characteristics of genomic regions more likely to have discordant variants which require additional care when making biological conclusions based on calls, or lack of calls, in these regions.

This evaluation work is part of a larger community effort to better characterize variant calling methods. A key component of these evaluations is a well characterized set of reference variations for the NA12878 human HapMap genome, provided by NIST’s Genome in a Bottle consortium. The diagnostic component of this work supplements emerging tools like GCAT (Genome Comparison and Analytic Testing), which provides a community platform for comparing and discussing calling approaches.

I’ll show a 12 way comparison between 2 different aligners (novoalign and bwa mem), 2 different post-alignment preparation methods (GATK best practices and the Marth lab’s gkno pipeline), and 3 different variant callers (GATK UnifiedGenotyper, GATK HaplotypeCaller, and FreeBayes). This allows comparison of openly available methods (bwa mem, gkno preparation, and FreeBayes) with those that require licensing (novoalign, GATK’s variant callers). I’ll also describe bcbio-nextgen, the fully automated open-source pipeline used for variant calling and evaluation, which allows others to easily bring this methodology into their own work and extend this analysis.

Aligner, post-alignment preparation and variant calling comparison

To compare methods, we called variants on a NA12878 exome dataset from EdgeBio’s clinical pipeline and assessed them against the NIST Genome in a Bottle reference material. Discordant positions where the reference and evaluation variants differ fall into three different categories:

  • Extra variants, called in the evaluation data but not in the reference. These are potential false positives.
  • Missing variants, found in the NA12878 reference but not in the evaluation data set. These are potential false negatives. The use of high quality reference materials from NIST enables identification of genomic regions we fail to call in.
  • Shared variants, called in both the evaluation and reference but differently represented. This could result from allele differences, such as heterozygote versus homozygote calls, or variant identification differences, such as indel start and end coordinates.

To further identify causes of discordance, we subdivide the missing and extra variants using annotations from the GEMINI variation framework:

We subdivide and restrict our comparisons to help identify sources of differences between methods indistinguishable when looking at total discordant counts. A critical subdivison is comparing SNPs and indels separately. With lower total counts of indels but higher error rates, each variant type needs independent visualization. Secondly, it’s crucial to distinguish between discordance caused by a lack of coverage, and discordance caused by an actual difference in variant assessment. We evaluate only in callable regions with 4 or more reads. This low minimum cutoff provides a valuable evaluation of low coverage regions, which differ the most between alignment and calling methods.

I’ll use this data to provide recommendations for alignment, post-alignment preparation and variant calling. In addition to these high level summaries, the full dataset and summary plots available below providing a starting place for digging further into the data.


We compared two recently released aligners designed to work with longer reads coming from new sequencing technologies: novoalign (3.00.02) and bwa mem (0.7.3a). bwa mem identified 1389 additional concordant SNPs and 145 indels not seen with novoalign. 1024 of these missing variants are in regions where novoalign does not provide sufficient coverage for calling. Of those, 92% (941) have low coverage with less than 10 reads in the bwa alignments. Algorithmic changes impact low coverage regions more due to the decreased evidence and susceptibility to crossing calling coverage thresholds, so we need extra care and consideration of calls in these regions.

Our standard workflow uses novoalign based on its stringency in resolving large insertions and deletions. These results suggest equally good results using bwa mem, along with improved processing times. One caveat to these results is that some of the available Illumina call data that feeds into NIST’s reference genomes comes from a bwa alignment, so some differences may reflect a bias towards bwa alignment heuristics. Using non-simulated reference data sets has the advantage of capturing real biological and process errors, but requires iterative improvement of the reference materials to avoid this type of potential algorithmic bias.

Comparison of concordant variants by aligner type

Post-alignment preparation and quality score recalibration

We compared two methods of quality recalibration:

  • GATK’s best practices (2.4-9): This involves de-duplication with Picard MarkDuplicates, GATK base quality score recalibration and GATK realignment around indels.
  • The Marth Lab’s gkno realignment pipeline: This performs de-duplication with samtools rmdup and realignment around indels using ogap. All commands in this pipeline work on streaming input, avoiding disk IO penalties by using unix pipes. This piped approach improves scaling on large numbers of whole genome samples. Notably, our implementation of the pipeline does not use a base quality score recalibration step.

GATK best practice pipelines offer an advantage over the gkno-only pipeline primarily because of improvements in SNP calling from base quality recalibration. Specifically we lose ~1% (824 / 77158) of called variations. These fall into the discordant missing “other” category, so we cannot explain them by metrics such as coverage or genome difficulty. The simplest explanation is that initial poor quality calculations in those regions result in callers missing those variants. Base quality recalibration helps recover them. These results match Brendan O’Fallon’s recent analysis of base quality score recalibration.

This places a practical number on the lost variants when avoiding recalibration either due to scaling or GATK licensing concerns. Some other options for recalibration include Novoalign’s Quality Recalibration and University of Michigan’s BamUtil recab, although we’ve not yet tested either in depth as potential supplements to improve calling in non-GATK pipelines.

Comparison of concordant variants by post-alignment prep method

Variant callers

For this comparison, we used three general purpose callers that handle SNPs and small indels, all of which have updated versions since our last comparison:

Adjusting variant calling methods has the biggest impact on the final set of calls. Called SNPs differ by 4577 between the three compared approaches, in comparison with aligner and post-alignment preparation changes which resulted in a maximum difference of 1389 calls. This suggests that experimenting with variant calling approaches currently provides the most leverage to improve calls.

A majority of the SNP concordance differences between the three calling methods are in low coverage regions with between 4 and 9 reads. GATK UnifiedGenotyper performs the best in detecting SNPs in these low coverage regions. FreeBayes and GATK HaplotypeCaller both call more conservatively in these regions, generating more potential false negatives. FreeBayes had the fewest heterozygote/homozygote discrimination differences of the three callers.

For indels, FreeBayes and HaplotypeCaller both provide improved sensitivity compared to UnifiedGenotyper, with HaplotypeCaller identifying the most, especially in low coverage regions. In contrast to the SNP calling results, FreeBayes has more calls that match the expected indel but differ in whether a call is a heterozygote or homozygote.

Comparison of concordant variants by calling method

No one caller outperformed the others on all subsets of the data. GATK UnifiedGenotyper performs best on SNPs but is less sensitive in resolving indels. GATK HaplotypeCaller identifies the most indels, but is more conservative than the other callers on SNPs. FreeBayes provides intermediate sensitivity and specificity between the two for both SNPs and indels. A combined UnifiedGenotyper and HaplotypeCaller pipeline for SNPs and indels, respectively, would provide the best overall calling metrics based on this set of comparisons.

Low coverage regions are the key area of difference between callers. Coupled with the alignment results and investigation of variant changes resulting from quality score binning, this suggests we should be more critical in assessing both calls and coverage in these regions. Assessing coverage and potential false negatives is especially critical since we lack good tools to summarize and prioritize genomic regions that are potentially missed during sequencing. This also emphasizes the role of population-based calling to help resolve low coverage regions, since callers can use evidence from multiple samples to better estimate the likelihoods of low coverage calls.

Automated calling and grading pipeline

Method comparisons become dated quickly due to the continuous improvement in aligners and variant callers. While these recommendations are useful now, in 6 months there will be new releases with improved approaches. This rapid development cycle creates challenges for biologists hoping to derive meaning from variant results: do you stay locked on software versions whose trade offs you understand, or do you attempt to stay current and handle re-verifying results with every new release?

Our goal is to provide a community developed pipeline and comparison framework that ameliorates this continuous struggle to re-verify. The analysis done here is fully automated as part of the bcbio-nextgen analysis framework. This framework code provides full exposure and revision tracking of all parameters used in analyses. For example, the ngsalign module contains the command lines used for bwa mem and novoalign, as well as all other tools.

To install the pipeline, third-party software and required data files:

python /usr/local /usr/local/share/bcbio-nextgen

The installer bootstraps all installation on a bare machine using the CloudBioLinux framework. More details and options are available in the installation documentation.

To re-run this analysis, retrieve the input data files and configuration as described in the bcbio-nextgen example documentation with:

$ mkdir config && cd config
$ wget\
$ cd .. && mkdir input && cd input
$ wget\
$ wget\
$ wget
$ wget
$ gunzip NA12878-nist-*.gz
$ wget
$ gunzip NGv3.bed.gz

Then run the analysis, distributed on 8 local cores, with:

$ mkdir work && cd work
$ bcbio_system.yaml ../input ../config/NA12878-exome-methodcmp.yaml -n 8

The bcbio-nextgen documentation describes how to parallelize processing over multiple machines using cluster schedulers (LSF, SGE, Torque).

The pipeline and comparison framework are open-source and configurable for multiple aligners, preparation methods and callers. We invite anyone interested in this work to provide feedback and contributions.

Full data sets

We extracted the conclusions for alignment, post-alignment preparation and variant calling from analysis of the full dataset. The visualizations for the full data are not as pretty but we make them available for anyone interested in digging deeper:

The comparison variant calls are also useful for pinpointing algorithmic differences between methods. Some useful subsets of variants:

  • Concordant variants called by bwa and not novoalign, where novoalign did not have sufficient coverage in the region. These are calls where either novoalign fails to map some reads, or bwa maps too aggressively: VCF of bwa calls with low or no coverage in novoalign.
  • Discordant variants called consistently by multiple calling methods. These are potential errors in the reference material, or consistently problematic calling regions for multiple algorithms. Of the 9004 shared discordants, the majority are potential false negatives not seen in the evaluation calls (7152; 79%). Another large portion is heterozygote/homozygote differences, which make up 1627 calls (18%). 6652 (74%) of the differences have low coverage in the exome evaluation, again reflecting the difficulties in calling in these regions. The VCF of discordants found in 2 or more callers contains these calls, with a ‘GradeCat’ INFO tag specifying the discordance category.

We encourage reanalysis and welcome suggestions for improving the presentation and conclusions in this post.

Written by Brad Chapman

May 6, 2013 at 8:29 am

The influence of reduced resolution quality scores on alignment and variant calling

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BAM file size reduction and quality score binning

We have a large upcoming whole genome sequencing project with Illumina, and they approached us about delivering BAM files with reduced resolution base quality scores. They have a white paper describing the approach, which involves binning scores to reduce resolution. This reduces the number of scores describing the quality of a base from 40 down to 8.

The advantage of this approach is a significant reduction in file size. BAM files use BGZF compression, and the underlying gzip DEFLATE algorithm compresses based on shared text regions. Reducing the number of quality values increases shared blocks and improves compression. This reduces BAM file sizes by 25-35%: an exome BAM file reduced from 5.7Gb to 3.7Gb after quality binning.

The potential downside is that the reduction in quality resolution may impact alignment and variant calling approaches that rely on base quality scores. To assess this, I implemented quality score binning as part of the bcbio-nextgen analysis pipeline using the CRAM toolkit and ran alignment, recalibration, realignment and variant calling on:

  • The original unbinned 40-resolution base quality BAM from an NA12878 exome.
  • The BAM binned into 8-resolution base qualities before alignment.
  • The BAM binned into 8-resolution base qualities before alignment and binned again following base quality score recalibration.

A comparison of alignment and variant calls from the three approaches indicates that binning has nearly no impact on alignment and a small impact on variant calls, primarily in low depth regions.

Alignment differences

We aligned 100bp paired end reads with Novoalign, a quality aware aligner. Comparison of mapped reads showed nearly no impact on total mapped reads. The plot below shows a generic delta of changes in mapped reads across the 22 autosomes alongside the increase in unmapped pairs. Out of 73 million total reads, the changes account for ~0.003% of the total reads. There also did not appear to be any worrisome patterns of loss for specific chromosomes. Overall, there is a minimal impact of quality score binning on the ability to align the reads.

Alignment changes following quality binning

Variant call differences

We called variants using the GATK Unified Genotyper following the best practice recommendations for exomes and then compared calls from original and binned quality scores. Both approaches for binning — pre-binning, and pre-binning plus post-quality recalibration binning — showed similar levels of concordance to non-binned quality scores: 99.81 and 99.78, respectively. Since the additional binning after recalibration provides a smaller prepared BAM file for storage and has a similar impact to pre-binning only, we used this for additional analysis of discordant variants.

The table below shows the discordant differences between the 40 quality score resolution and binned, 8 quality score resolution BAMs. 40 quality discordant variants are those called with full quality score resolution but not called, or called differently, after binning to 8 quality score resolution. Conversely, the 8-quality discordants are those called uniquely after quality binning:

Overall genotype concordance 99.78
concordant: total 117887
concordant: SNPs 109144
concordant: indels 8743
40-quality discordant: total 821
40-quality discordant: SNPs 759
40-quality discordant: indels 62
8-quality discordant: total 1289
8-quality discordant: SNPs 1240
8-quality discordant: indels 49
het/hom discordant 259

We investigated the discordant variants further since 1.5% of the total variant calls change as a result of binning, Of the 1851 unique discordant variants, approximately half (928) fall into reproducible variants identified by looking at ensemble combinations of replicates. Of these potentially problematic discordant variants more than half are in low coverage regions with less than 10 reads:

Variant changes following quality binning

The major influence of quality score binning is resolution of variants in low coverage regions. This manifests as differences in heterozygote and homozygote calling, indel representation and filtering differences related to quality and mappability. To assess the potential impact, we looked at the loss in callable bases on a 30x whole genome sequence when moving from a minimum of 5 reads to a minimum of 10, using GATK’s CallableLoci tool. Regions with read coverage of 5 to 9 make up 4.7 million genome positions, 0.17% of the total callable bases.

5 read minimum 10 read minimum
Callable bases 2,775,871,235 2,771,109,000
Percent callable 96.90% 96.73%
Low coverage 17,641,980 22,404,215
No coverage/ poor mapping 71,272,008 71,272,008

In conclusion, quality score binning provides a useful reduction in input file sizes with minimal impact on alignment. For variant calling, use additional caution in low coverage regions with less than 10 supporting reads. Given the rapid increases in read throughput that are driving the need for file size reduction, quality score binning is a worthwhile tradeoff for high-coverage recalling work.

Written by Brad Chapman

February 13, 2013 at 5:49 am

Posted in variation

Tagged with , , ,