Contaminant DNA in bacterial sequencing experiments is a major source of false genetic variability

Background Contaminant DNA is a well-known confounding factor in molecular biology and in genomic repositories. Strikingly, analysis workflows for whole-genome sequencing (WGS) data commonly do not account for errors potentially introduced by contamination, which could lead to the wrong assessment of allele frequency both in basic and clinical research. Results We used a taxonomic filter to remove contaminant reads from more than 4000 bacterial samples from 20 different studies and performed a comprehensive evaluation of the extent and impact of contaminant DNA in WGS. We found that contamination is pervasive and can introduce large biases in variant analysis. We showed that these biases can result in hundreds of false positive and negative SNPs, even for samples with slight contamination. Studies investigating complex biological traits from sequencing data can be completely biased if contamination is neglected during the bioinformatic analysis, and we demonstrate that removing contaminant reads with a taxonomic classifier permits more accurate variant calling. We used both real and simulated data to evaluate and implement reliable, contamination-aware analysis pipelines. Conclusion As sequencing technologies consolidate as precision tools that are increasingly adopted in the research and clinical context, our results urge for the implementation of contamination-aware analysis pipelines. Taxonomic classifiers are a powerful tool to implement such pipelines.