title: Diverse and widespread contamination evident in the unmapped depths of high throughput sequencing data.
abstract: Trace quantities of contaminating DNA are widespread in the laboratory environment, but their presence has received little attention in the context of high throughput sequencing. This issue is highlighted by recent works that have rested controversial claims upon sequencing data that appear to support the presence of unexpected exogenous species. I used reads that preferentially aligned to alternate genomes to infer the distribution of potential contaminant species in a set of independent sequencing experiments. I confirmed that dilute samples are more exposed to contaminating DNA, and, focusing on four single-cell sequencing experiments, found that these contaminants appear to originate from a wide diversity of clades. Although negative control libraries prepared from 'blank' samples recovered the highest-frequency contaminants, low-frequency contaminants, which appeared to make heterogeneous contributions to samples prepared in parallel within a single experiment, were not well controlled for. I used these results to show that, despite heavy replication and plausible controls, contamination can explain all of the observations used to support a recent claim that complete genes pass from food to human blood. Contamination must be considered a potential source of signals of exogenous species in sequencing data, even if these signals are replicated in independent experiments, vary across conditions, or indicate a species which seems a priori unlikely to contaminate. Negative control libraries processed in parallel are essential to control for contaminant DNAs, but their limited ability to recover low-frequency contaminants must be recognized.
authors.affiliation: Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, Michigan, United States of America.
authors.forename: Richard W
authors.initials: RW
authors.lastname: Lusk
mesh_terms: D059018:DNA Contamination; D004269:DNA, Bacterial / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing / Q000592:standards*; D006801:Humans; D017422:Sequence Analysis, DNA / Q000592:standards*
chemical_list: D004269:DNA, Bacterial