Workflow: 16S sequencing

Performing accurate species-level bacterial identification with nanopore sequencing

16S sequencing is the predominant method for microbial identification and has a wide range of applications, including food safety, environmental and conservation monitoring, pathogen detection, and clinical microbiology. The 16S ribosomal RNA (rRNA) gene is ~1.5 kb and comprised of nine variable regions divided by highly conserved sequences1. Using legacy sequencing technology, species-level bacterial identification is challenging because the short reads cannot span the full gene, limiting resolution. Instead, only partial fragments of the gene are sequenced, for example the V3–V4 or V4–V5 regions. However, nanopore technology can overcome these limitations by generating long reads spanning V1–V9 regions of the 16S rRNA gene in a single read. By sequencing the entire gene rather than subsets of exons, greater taxonomic resolution is achieved for accurate species identification from polymicrobial samples. In this targeted workflow, the 16S rRNA gene is first amplified by PCR with 16S primers and then sequenced with long nanopore reads, providing a rapid and cost-effective method of species-level microbial identification.

Here we present a rapid workflow for full-length 16S rRNA sequencing of polymicrobial samples, using MinION™ Flow Cells on a MinION or GridION™ sequencing device and an EPI2ME™ analysis solution.