The GATK joint genotyping workflow is appropriate for calling variants in RNA-seq experiments

Area of Science:

• Genomics
• Bioinformatics
• Molecular Biology

Background:

• The Genome Analysis Toolkit (GATK) is widely used for variant discovery in next-generation sequencing data.
• Current GATK recommendations for RNA sequencing (RNA-seq) involve per-sample variant calling, which has limitations in reporting genotypes.
• GATK versions 3.0+ enable cohort analysis using Genomic Variant Call Format (GVCF) mode for improved variant calling.

Purpose of the Study:

• To evaluate the GATK joint genotyping method using GVCF mode for variant calling in RNA-seq data.
• To compare the performance of GATK joint genotyping against a per-sample variant calling approach for RNA-seq.
• To assess the sensitivity, precision, and accuracy of both methods using DNA genotypes as a reference.

Main Methods:

• RNA-seq data from 50 cows' primary macrophages were analyzed.
• Variant calling was performed using GATK's joint genotyping (GVCF mode) and per-sample methods.
• Pair-wise comparisons were conducted against DNA genotypes from genotyping-by-sequencing and Bovine SNP50 Beadchip.

Main Results:

• Both GATK methods showed similar performance in detecting reference variants.
• The joint genotyping method demonstrated higher sensitivity than the per-sample method for RNA-seq variant calling.
• The joint genotyping approach offers greater flexibility and technical ease.

Conclusions:

• The GATK joint genotyping method is a recommended approach for variant calling in RNA-seq experiments.
• This method provides advantages over per-sample calling, including reporting homozygous reference genotypes and missing data.
• The findings support the adoption of GVCF-based joint genotyping for enhanced RNA-seq variant discovery.