Single‐cell genomics: unravelling the genomes of unculturable microorganisms

Abstract

Microbial genomics and related transcriptomics methods rely on culturing techniques to obtain enough DNA suitable for high‐throughput sequencing without resorting to DNA amplification techniques. A few microgram of DNA is needed for most common next‐generation sequencing methods. For transcriptome analysis, sufficient cDNA is needed to measure low abundance mRNA copies in the cell. However, the large majority of microbes on earth resist cultivation, hampering research into their relevant gene pool, ecological niche or industrial relevance. For example, many environmental or gut‐related species cannot be grown outside their natural habitat. Even if we isolate the metagenome or the metatranscriptome from these environments, this reveals only a fragmented sequence landscape that is difficult to assign to individual species. Although enrichment techniques or metatransciptome analysis of previously unculturable species have been shown to assist in directed culturing, e.g. of a Rikenella‐like bacterium (Bomar et al., 2011), the unravelling of a complex metagenome into its individual genomes and their organization is impossible using current technologies. A major challenge is the analysis of bacteria and other organisms living inside a complex matrix, like biofilms. Metagenome or transcriptome analysis of microorganisms has been described for biofilms consisting of a single species by scraping of the biofilm to obtain enough material (Holmes et al., 2006), but for multi‐species biofilms this method results in a metagenome or metatranscriptome dataset. The solution to these challenges may be the isolation and genomic analysis of unculturable single cells isolated from such environments. Here we describe in brief the state‐of‐the‐art in single‐cell microbial genomics.