Massively parallel sequencing of single cells by epicPCR links functional genes with phylogenetic markers

ISME J. 2016 Feb;10(2):427-36. doi: 10.1038/ismej.2015.124. Epub 2015 Sep 22.

Abstract

Many microbial communities are characterized by high genetic diversity. 16S ribosomal RNA sequencing can determine community members, and metagenomics can determine the functional diversity, but resolving the functional role of individual cells in high throughput remains an unsolved challenge. Here, we describe epicPCR (Emulsion, Paired Isolation and Concatenation PCR), a new technique that links functional genes and phylogenetic markers in uncultured single cells, providing a throughput of hundreds of thousands of cells with costs comparable to one genomic library preparation. We demonstrate the utility of our technique in a natural environment by profiling a sulfate-reducing community in a freshwater lake, revealing both known sulfate reducers and discovering new putative sulfate reducers. Our method is adaptable to any conserved genetic trait and translates genetic associations from diverse microbial samples into a sequencing library that answers targeted ecological questions. Potential applications include identifying functional community members, tracing horizontal gene transfer networks and mapping ecological interactions between microbial cells.

Publication types

  • Evaluation Study
  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Bacteria / classification*
  • Bacteria / genetics
  • Bacteria / isolation & purification*
  • Bacterial Proteins / genetics
  • DNA, Bacterial / genetics
  • High-Throughput Nucleotide Sequencing / methods*
  • Lakes / microbiology*
  • Metagenomics
  • Molecular Sequence Data
  • Phylogeny*
  • RNA, Ribosomal, 16S / genetics

Substances

  • Bacterial Proteins
  • DNA, Bacterial
  • RNA, Ribosomal, 16S