Single-cell genomics of microbial dark matter

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

Virtual microfluidics for digital quantification and single-cell sequencing

We have developed hydrogel-based virtual microfluidics as a simple and robust alternative to complex engineered microfluidic systems for the compartmentalization of nucleic acid amplification reactions. We applied in-gel digital multiple displacement amplification (dMDA) to purified DNA templates, cultured bacterial cells and human microbiome samples in the virtual microfluidics system, and demonstrated whole-genome sequencing of single-cell MDA products with excellent coverage uniformity and markedly reduced chimerism compared with products of liquid MDA reactions.Broad Institute of MIT and Harvard (Lawrence Summers Fellowship)Burroughs Wellcome Fund (Career Award)Massachusetts Institute of Technology. Center for Microbiome Informatics and TherapeuticsNational Human Genome Research Institute (U.S.) (Broad Institute of MIT and Harvard. Grant U54HG003067)Massachusetts Institute of Technology. Center for Environmental Health SciencesFiji. Ministry of Healt