Characterizing Microbiomes via Sequencing of Marker Loci: Techniques To Improve Throughput, Account for Cross-Contamination, and Reduce Cost

Abstract

New methods to characterize microbiomes reduce technology-imposed limitations to study design, but many new approaches have not been widely adopted. Here, we present techniques to increase throughput and reduce contamination alongside a thorough review of current best practices.New approaches to characterizing microbiomes via high-throughput sequencing provide impressive gains in efficiency and cost reduction compared to approaches that were standard just a few years ago. However, the speed of method development has been such that staying abreast of the latest technological advances is challenging. Moreover, shifting laboratory protocols to include new methods can be expensive and time consuming. To facilitate adoption of new techniques, we provide a guide and review of recent advances that are relevant for single-locus sequence-based study of microbiomes—from extraction to library preparation—including a primer regarding the use of liquid-handling automation in small-scale academic settings. Additionally, we describe several amendments to published techniques to improve throughput, track contamination, and reduce cost. Notably, we suggest adding synthetic DNA molecules to each sample during nucleic acid extraction, thus providing a method of documenting incidences of cross-contamination. We also describe a dual-indexing scheme for Illumina sequencers that allows multiplexing of many thousands of samples with minimal PhiX input. Collectively, the techniques that we describe demonstrate that laboratory technology need not impose strict limitations on the scale of molecular microbial ecology studies. IMPORTANCE New methods to characterize microbiomes reduce technology-imposed limitations to study design, but many new approaches have not been widely adopted. Here, we present techniques to increase throughput and reduce contamination alongside a thorough review of current best practices.