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.

Development of a Bench-Top Extra-Cleanroom for DNA Amplification

“…The WGA reaction was per formed in triplicate on separate days in three different laboratories (Hiroshima University (HU), NFRI, and the Isehara Research Lab (IRL) of Kanto Chemical Co., Inc) under ISO-5 conditions using the same DNA-free reagents (Supplementary Figure S4). As observed previously (18), sequencing of DNA amplified in the NTCs showed that DNA contamination under ISO-5 conditions was derived from various sources, regardless of the laboratory (Supplementary Tables S3S5). In addition, it was confirmed that the reagents used in these experiments were DNA-free because amplicons were not found in one set of NTCs (Day 3 in IRL; Supplementary Figure S4).…”

“…Furthermore, two recent reports have shown that contaminating DNA can also be found in high-throughput next-generation sequencing (NGS) reads (18, 19). Considering that DNA contamination could randomly occur in different tubes during preparation of the WGA reaction mixture, the DNA sequences of the NTC amplicons determined by NGS could not be used for the removal of contaminating sequences from target sequence read data of WGA samples.…”

Virome Assembly and Annotation: A Surprise in the Namib Desert

“…Yet, these procedures can introduce biases such as selective purification of DNA from specific organisms, DNA fractionation and selective sequence amplification (Kim and Bae, 2011; Yuan et al, 2012; van Dijk et al, 2014) that will affect representation of the viral and non-viral DNA. Lower target/contaminant DNA ratios result in higher numbers of reads from contaminants in the read dataset, as demonstrated by Lusk (2014), who found that frequencies of contaminating human reads in an E. coli study increased with decreasing sample DNA concentrations. In this study, we followed all standard laboratorial practices for virome analyses, including sterile handling of material, microscopic examination of the sample for bacterial cells, TEM to verify presence of viral particles, as well as DNAse and RNAse treatment of the viral particles prior to protein coat digestion (verified through 16S rRNA PCR with positive and negative controls).…”

Mosquito vector‐associated microbiota: Metabarcoding bacteria and eukaryotic symbionts across habitat types in Thailand endemic for dengue and other arthropod‐borne diseases

“…To limit biases introduced by PCR, we performed a strict sequence quality control. In the future, the use of mock community control, although not common practice at the time of this study, is recommended to quantify sequence contamination (Lusk, 2014; Salter et al., 2014). However, the latter study also show that contamination is more pronounced with samples containing low microbial biomass.…”