On the impact of reference selection on variant calling in phylogenomics: How to avoid systematic error in target-enrichment studies of non-model organisms

Abstract

Target-enrichment methods are widely used in phylogenomic studies aiming to reconstruct the relationships among diverse groups of organisms. When building phylogenomic datasets from short sequencer reads, software pipelines need to accurately align short sequencer reads to the target and identify the genetic variation in homologous DNA sequences. Variant calling, a crucial step in this process, is known to be error-prone in a phylogenetic analysis framework as it requires a conspecific reference as a template. To avoid problems relating to reference choice, existing target-enrichment pipelines often skip variant calling entirely using work-arounds. This study investigates the effect of reference genome selection on the reliability of variant calling and assesses the consequences of reference genome choice on phylogenomic analyses downstream. Employing an empirical exon capture sequence dataset, we examine how variant detection is influenced by reference genome choice. We reconstructed and analysed phylogenomic sequence datasets from short sequencer reads, which includes steps of quality control, read mapping, variant calling, multiple sequence alignment and phylogenetic analysis. We assembled the same exon capture dataset by using four different references for variant calling. These references were (1) a distantly related species, (2) a single ingroup sample, (3) a composite of all ingroup samples, and (4) a self-derived reference for each sample. We found that reference choice significantly impacted the variant detection and that these differences in variant detection influenced the phylogenetic reconstructions downstream. Comparing multiple sequence alignments and phylogenetic trees produced using different references revealed that employing a sample-specific self-reference for variant calling maximizes the accuracy of the variant detection process. Based on this finding, we recommend incorporating self-referenced variant calling in the assembly of phylogenomic datasets to ensure robustness and reproducibility, especially for datasets characterized by low read coverage.