ChoCallate: a computational pipeline for consensus germline variant calling in genomic sequencing data

Accurate germline variant calling in non-model and polyploid organisms remains challenging due to sequencing errors, genomic complexity, and caller-specific biases. Existing ensemble methods of variant calling are constrained by species-specific training, limited portability, or an inability to process multiple samples concurrently. To address these limitations we developed ChoCallate, a Nextflow-based pipeline for consensus germline variant calling. The pipeline integrates three species-independent callers, bcftools, FreeBayes, and Genome Analysis Toolkit (GATK) HaplotypeCaller, within a flexible voting mechanism. A distinctive feature of ChoCallate is its incorporation of coverage-aware consensus generation via Browser Extensible Data (BED) file, enabling reliable variant identification even in reduced-representation sequencing data such as Restriction site Associated DNA sequencing (RAD-seq) or RNA-seq. In a simulated dataset containing 10,000 single-nucleotide polymorphisms (SNPs) and 1,000 InDels of Arabidopsis thaliana across coverage depths ranging from 3× to 50×, the two-vote consensus strategy provided a favourable precision-recall balance, particularly at lower coverage (3×–10×), where SNP precision was enhanced without substantial loss of sensitivity. False-negative variants were found to concentrate in centromeric and nucleolus organizer regions (NORs) across all strategies, indicating that repetitive genomic structure, rather than consensus logic, constitutes the primary limitation. In a case study of 200 bread wheat ( Triticum aestivum ) samples, ChoCallate identified 11,178 high-confidence variants, and a genome-wide association study (GWAS) for awnedness successfully recovered the known B1/ALI-1 locus on chromosome 5A, replicating prior findings with reduced genomic inflation ( λ (gc) = 1.06) by decreasing the number of false-positive variants through a consensus-based approach. ChoCallate is portable, species-agnostic, and freely available.