Negligible effects of read trimming on the accuracy of germline short variant calling in the human genome

Abstract

Next generation sequencing (NGS) has become a standard tool in the molecular diagnostics of Mendelian disease, and the precision of such diagnostics is greatly affected by the accuracy of variant calling from sequencing data. Recently, we have made a comprehensive evaluation of the performance of multiple variant calling pipelines, showing that state-of-the-art neural network-based methods show the best accuracy of variant discovery in the coding genome. In this work, we systematically evaluated the effects of adapters on the performance of variant calling tools using standard reference Genome-in-a-Bottle (GIAB) samples. We show that adapter trimming has no effect on the accuracy of the best-performing variant callers (e.g., DeepVariant) on whole-genome sequencing (WGS) data. For whole-exome sequencing (WES) datasets subtle improvement of accuracy was observed in some of the samples. In high-coverage WES data ([~]200x mean coverage), adapter removal allowed for discovery of 2-4 additional true positive variants in only two out of seven datasets tested. Moreover, this effect was not dependent on the median insert size and proportion of adapter sequences in reads. Surprisingly, the effect of trimming on variant calling was reversed when moderate coverage ([~]80-100x) WES data was used. Finally, we show that some of the recently developed machine learning-based variant callers demonstrate greater dependence on the presence of adapters in reads. Taken together, our results indicate that adapter removal is unnecessary when calling germline variants, but suggest that preprocessing methods should be carefully chosen when developing and using machine learning-based variant analysis methods.