GATK hard filtering: tunable parameters to improve variant calling for next generation sequencing targeted gene panel data

Abstract

BackgroundNGS technology represents a powerful alternative to the standard Sanger sequencing in the context of clinical setting. The proprietary software that are generally used for variant calling often depend on preset parameters that may not fit in a satisfactory manner for different genes.GATK, which is widely used in the academic world, is rich in parameters for variant calling. However the self-adjusting parameter calibration of GATK requires data from a large number of exomes. When these are not available, which is the standard condition of a diagnostic laboratory, the parameters must be set by the operator (hard filtering). The aim of the present paper was to set up a procedure to assess the best parameters to be used in the hard filtering of GATK. This was pursued by using classification trees on true and false variants from simulated sequences of a real dataset data.ResultsWe simulated two datasets, with different coverages, including all the sequence alterations identified in a real dataset according to their observed frequencies. Simulated sequences were aligned with standard protocols and then regression trees were built up to identify the most reliable parameters and cutoff values to discriminate true and false variant calls. Moreover, we analyzed flanking sequences of region presenting a high rate of false positive calls observing that such sequences present a low complexity make up.ConclusionsOur results showed that GATK hard filtering parameter values can be tailored through a simulation study based-on the DNA region of interest to ameliorate the accuracy of the variant calling.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-017-1537-8) contains supplementary material, which is available to authorized users.

Metagenome analysis of Russian and Georgian Pyophage cocktails and a placebo‐controlled safety trial of single phage versus phage cocktail in healthy Staphylococcus aureus carriers

“…Variants to detect single nucleotide polymorphisms (SNPs) were called using Haplotype from the Genome Analysis Toolkit (GATC) with a sample ploidy of 3 (McKenna et al, 2010). Variants were hard filtered using GATC recommended parameters with an additional filter for allelic frequency (AF < 0.1) and genotype quality (GQ < 30.0) (Summa et al, 2017). Local nucleotide alignments were done with Mummer v4.0beta package (nucmer -c 100 -L 100).…”

Comparative genome and transcriptome analysis unravels key factors of nitrogen use efficiency in Brassica napus L

“…The SAMtools (v 1.4) software (Li, 2011) was used to estimate coverage of the reference genome. The filtered alignment files were used for the identification of single nucleotide polymorphisms (SNPs) and Insertion/Deletions (InDels) using the Genome Analysis Toolkit (GATK, v3.6) software (De Summa et al, 2017). The CNVnator software (v 0.1.18) (Abyzov, Urban, Snyder, & Gerstein, 2011) was utilized to detect copy number variations (CNVs).…”

Bioinformatics workflows for genomic analysis of tumors from Patient Derived Xenografts (PDX): challenges and guidelines

“…While the default thresholds for GATK hard filtering parameters [29] removed a large proportion of the FPs, applying Xenome to filter for human reads yielded superior performance in terms of substantially higher precision, as well as improvement in recall. In addition, Xenome filtering maintained the correlation between the predicted versus actual allele frequencies, which would otherwise decrease with higher mouse contamination ( Supplementary Table S2).…”

“…Presence of alternate loci in the genome assembly. The GRCh38.p5 human genome assembly includes 262 regions of alternate loci to account for human chromosomal regions that exhibit sufficient variability to prevent adequate representation by a single sequence [29]. As such, we aligned the reads to both primary and alternate chromosomal reference sequences using BWA-MEM with ALT-aware.…”