A Lot on Your Plate? Well-to-Well Contamination as an Additional Confounder in Microbiome Sequence Analyses.

Alan W Walker

doi:10.1128/msystems.00362-19

Abstract

DNA sequence-based microbiome studies can be impacted by a range of different methodological artefacts. Contamination originating from laboratory kits and reagents can lead to erroneous results, particularly in samples containing a low microbial biomass. Minich and colleagues (mSystems 4:e00186-19, 2019, https://doi.org/10.1128/mSystems.00186-19) report on a different form of contamination, cross-contamination between samples that are processed together. They find that transfer of material between samples in 96-well plates is a common occurrence. The DNA extraction step, particularly when carried out automatedly, is identified as the major source of this contamination type. Well-to-well contamination distorts diversity measures, with low-biomass samples particularly affected. This report has important implications for attempts to decontaminate microbiome sequencing results. As contamination is derived from both external sources and crossover between samples, it is not appropriate to simply remove sequence variants that are detected in negative-control blanks, and more-nuanced decontamination approaches may be required.

Highlights

DNA sequence-based microbiome studies can be impacted by a range of different methodological artefacts
The last 15 years have seen a rapid escalation in research on microbial communities, largely driven by the advent and widespread adoption of high-throughput sequencing techniques [1, 2]
A further potential problem with DNA sequence-based microbiome profiling methods is contamination, as the laboratory kits and reagents that are used to process samples for subsequent sequencing are not sterile. Contamination arising from these sources was first reported in the 1990s [6], and more recent work has demonstrated the impact that these contaminants can have on modern microbiome profiling studies [7,8,9,10,11,12]

Summary

Introduction

DNA sequence-based microbiome studies can be impacted by a range of different methodological artefacts. While it has been an incredibly productive and exciting time for microbiome research, some of the results generated using sequence-based approaches have been controversial and occasionally contradict conventional knowledge. A further potential problem with DNA sequence-based microbiome profiling methods is contamination, as the laboratory kits and reagents that are used to process samples for subsequent sequencing are not sterile.

Results

Conclusion