Improved genome recovery and integrated cell-size analyses of individual uncultured microbial cells and viral particles
July 20, 2017
Cell sorting, lysis, and gDNA amplification setup were performed in a HEPAfiltered environment conforming to Class 1000 cleanroom specifications. Prior to cell sorting, the instrument, the reagents, and the workspace were decontaminated for DNA using UV irradiation and sodium hypochlorite solution, as previously described61. To further reduce the risk of DNA contamination, and to improve accuracy and throughput, Bravo (Agilent Technologies) and Freedom Evo (Tecan) robotic liquid handlers were used for all liquid handling in 384-well plates.
Microbial single-cell genomics can be used to provide insights into the metabolic potential, interactions, and evolution of uncultured microorganisms. Here we present WGA-X, a method based on multiple displacement amplification of DNA that utilizes a thermostable mutant of the phi29 polymerase. WGA-X enhances genome recovery from individual microbial cells and viral particles while maintaining ease of use and scalability. The greatest improvements are observed when amplifying high G+C content templates, such as those belonging to the predominant bacteria in agricultural soils. By integrating WGA-X with calibrated index-cell sorting and high-throughput genomic sequencing, we are able to analyze genomic sequences and cell sizes of hundreds of individual, uncultured bacteria, archaea, protists, and viral particles, obtained directly from marine and soil samples, in a single experiment. This approach may find diverse applications in microbiology and in biomedical and forensic studies of humans and other multicellular organisms.Single-cell genomics can be used to study uncultured microorganisms. Here, Stepanauskas et al. present a method combining improved multiple displacement amplification and FACS, to obtain genomic sequences and cell size information from uncultivated microbial cells and viral particles in environmental samples.
Stepanauskas, R; Fergusson, EA; Brown, J; Poulton, NJ; Tupper, B; Labonté, JM; Becraft, ED; Brown, JM; Pachiadaki, MG; Povilaitis, T; Thompson, BP; Mascena, CJ; Bellows, WK; Lubys, A;
Journal: Nat Commun
Original article (28729688)