Single-Cell Genomics of Microbial Dark Matter

Methods Mol Biol. 2018:1849:99-111. doi: 10.1007/978-1-4939-8728-3_7.

Abstract

Single-cell genomics allows bypassing the culturing step and to directly access environmental microbes one cell at a time. The method has been successfully applied to explore archaeal and bacterial candidate phyla, referred to as microbial dark matter. Here I summarize the single-cell genomics workflow, including sample preparation and preservation, high-throughput fluorescence-activated cell sorting, cell lysis and amplification of environmental samples. Furthermore I describe phylogenetic screening based on 16S rRNA genes and suggest a suitable library preparation and sequencing approach.

Keywords: 16S rRNA gene; Biofilm; FACS; Fluorescence-activated cell sorting; ILLUMINA Nextera XT libraries; Microbial dark matter; Multiple genome amplification; Sediment; Single-cell genomics; Sludge.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Archaea / classification*
  • Archaea / genetics
  • Archaea / isolation & purification
  • Bacteria / classification*
  • Bacteria / genetics
  • Bacteria / isolation & purification
  • Flow Cytometry / methods*
  • Gene Library
  • Genomics / methods*
  • High-Throughput Nucleotide Sequencing / methods*
  • RNA, Ribosomal, 16S / genetics*
  • Single-Cell Analysis / methods*

Substances

  • RNA, Ribosomal, 16S