Detailed comparison of two popular variant calling packages for exome and targeted exon studies

Abstract

The Genome Analysis Toolkit (GATK) is commonly used for variant calling of single nucleotide polymorphisms (SNPs) and small insertions and deletions (indels) from short-read sequencing data aligned against a reference genome. There have been a number of variant calling comparisons against GATK, but an equally comprehensive comparison for VarScan not yet been performed. More specifically, we compare (1) the effects of different pre-processing steps prior to variant calling with both GATK and VarScan, (2) VarScan variants called with increasingly conservative parameters, and (3) filtered and unfiltered GATK variant calls (for both the UnifiedGenotyper and the HaplotypeCaller). Variant calling was performed on three datasets (1 targeted exon dataset and 2 exome datasets), each with approximately a dozen subjects. In most cases, pre-processing steps (e.g., indel realignment and quality score base recalibration using GATK) had only a modest impact on the variant calls, but the importance of the pre-processing steps varied between datasets and variant callers. Based upon concordance statistics presented in this study, we recommend GATK users focus on “high-quality” GATK variants by filtering out variants flagged as low-quality. We also found that running VarScan with a conservative set of parameters (referred to as “VarScan-Cons”) resulted in a reproducible list of variants, with high concordance (>97%) to high-quality variants called by the GATK UnifiedGenotyper and HaplotypeCaller. These conservative parameters result in decreased sensitivity, but the VarScan-Cons variant list could still recover 84–88% of the high-quality GATK SNPs in the exome datasets. This study also provides limited evidence that VarScan-Cons has a decreased false positive rate among novel variants (relative to high-quality GATK SNPs) and that the GATK HaplotypeCaller has an increased false positive rate for indels (relative to VarScan-Cons and high-quality GATK UnifiedGenotyper indels). More broadly, we believe the metrics used for comparison in this study can be useful in assessing the quality of variant calls in the context of a specific experimental design. As an example, a limited number of variant calling comparisons are also performed on two additional variant callers.

Keywords

• Variant calling
• Exome
• Targeted sequencing
• GATK
• VarScan
• SNP