Ubiquitous microbial contaminants associated with scientific ocean drilling

Deep-sea subsurface samples typically have low microbial biomass, making them more susceptible to contamination. Potential contaminants can be introduced during any step of the scientific process, including drilling (contamination from the drilling fluid and lubricants and seawater); sample preparation (contamination from air and human handling); and DNA extraction, amplification and sequencing (contamination from reagents). The International Ocean Discovery Program (IODP) samples that are dedicated to microbiological analyses (known as MBIO samples) are routinely tested for contamination by injecting known concentrations of fluorescent microspheres or tracers directly into the drilling fluids. These tracers are a great tool to determine the level of drilling fluid contamination on board, but they are not consistently used, and contamination of the samples could occur during other steps of sample processing and analysis. Moreover, there is an increased risk of contamination dominating the results of microbial surveys using PCR amplification of marker genes. Here, we built a database of common contaminants through the screening of contamination controls from available 16S rRNA gene amplicon datasets from past IODP expeditions. These controls included various lubricants used on board, drilling fluids, seawater, DNA extraction blanks and PCR blanks. The order Burkholderiales dominated most of the very low biomass samples, including negative controls, indicating the order's ubiquity and its potential to be overamplified with common 16S rRNA amplification protocols. We amplified the 16S rRNA gene from preserved IODP legacy microbiological core samples and tested their level of contamination using the database. We also looked at published studies that did not sequence negative controls. Our results demonstrate that the type of drilling, amount of manipulation of the sample prior to preservation and sample depth, often associated with biomass, can influence the level of contamination within subsurface samples. This work provides an analysis framework for microbial taxonomic survey studies from low biomass subsurface samples for future scientific ocean drilling expeditions.