Generating amplicon reads for microbial community assessment with next-generation sequencing

Marker gene amplicon sequencing is often preferred over whole genome sequencing for microbial community characterization, due to its lower cost while still enabling assessment of uncultivable organisms. This technique involves many experimental steps, each of which can be a source of errors and bias. We present an up-to-date overview of the whole experimental pipeline, from sampling to sequencing reads, and give information allowing for informed choices at each step of both planning and execution of a microbial community assessment study. When applicable, we also suggest ways of avoiding inherent pitfalls in amplicon sequencing.

16S rRNA gene; error and bias sources; experimental workflow; marker gene amplicons; microbial community assessment; next-generation sequencing.