Next-generation sequencing: insights to advance clinical investigations of the microbiome

Abstract

Next-generation sequencing (NGS) technology has advanced our understanding of the human microbiome by allowing for the discovery and characterization of unculturable microbes with prediction of their function. Key NGS methods include 16S rRNA gene sequencing, shotgun metagenomic sequencing, and RNA sequencing. The choice of which NGS methodology to pursue for a given purpose is often unclear for clinicians and researchers. In this Review, we describe the fundamentals of NGS, with a focus on 16S rRNA and shotgun metagenomic sequencing. We also discuss pros and cons of each methodology as well as important concepts in data variability, study design, and clinical metadata collection. We further present examples of how NGS studies of the human microbiome have advanced our understanding of human disease pathophysiology across diverse clinical contexts, including the development of diagnostics and therapeutics. Finally, we share insights as to how NGS might further be integrated into and advance microbiome research and clinical care in the coming years.

Figure 1. Bacterial 16S rRNA gene.

(A) Percentage sequence identity of conserved and hypervariable regions of the bacterial 16S rRNA gene. Adapted with permission from the Journal of Microbiological Methods (17) and Ilona Lehtinen (137). (B) Illustration of conserved and hypervariable regions corresponding to A and PCR amplification of the V1–V3 region of the bacterial 16S rRNA gene. Adapted with permission from Humana Press (148). (C) Schematic of 16S rRNA gene structure with hypervariable regions (V1–V9) labeled.

Figure 2. NGS implementation.

Overview of key steps in 16S rRNA gene sequencing, shotgun metagenomic sequencing, and RNA sequencing processes. ¹Host DNA or RNA depletion can be performed (optional steps). ²PCR amplification is used to amplify bacterial 16S rRNA gene variable regions (16S rRNA amplicon sequencing) or random cDNA fragments resulting from RNA reverse transcription for RNA sequencing. DNA-based shotgun metagenomic sequencing is optimally done without use of PCR amplification to avoid introduction of PCR-associated experimental bias. However, in samples with low DNA quantities, PCR amplification of the DNA library is sometimes used. ³Commonly Illumina-based sequencing chemistry (33). 4The taxonomic and functional analyses of NGS data are complex and make use, most often, of software available in the public domain.