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Recent advances in genomic DNA sequencing of microbial species from single cells

Abstract

The vast majority of microbial species remain uncultivated and, until recently, about half of all known bacterial phyla were identified only from their 16S ribosomal RNA gene sequence. With the advent of single-cell sequencing, genomes of uncultivated species are rapidly filling in unsequenced branches of the microbial phylogenetic tree. The wealth of new insights gained from these previously inaccessible groups is providing a deeper understanding of their basic biology, taxonomy and evolution, as well as their diverse roles in environmental ecosystems and human health.

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Figure 1: Complementary methods used to investigate the genomics of uncultivated bacteria.
Figure 2: Filling in the bacterial tree of life.
Figure 3: Comparative genomics using single-cell DNA amplification.

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Acknowledgements

The authors acknowledge discussions with G. Tesler, S. Yooseph and J. Badger. They also acknowledge assistance with the phylogenetic tree from C. Rinke and T. Woyke. This work was supported by grants to R.S.L. from the Alfred P. Sloan Foundation (Sloan Foundation-2007-10-19) and the US National Institutes of Health (NIH 2R01 HG003647 and NIH-HHSN272200900007C), and by grants to J.S.M. from the US National Institute of General Medical Sciences (NIH 1R01GM095373).

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Correspondence to Roger S. Lasken.

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Glossary

16S ribosomal RNA gene PCR analysis

A method in which primers designed for highly conserved regions of the 16S rRNA gene enable PCR from most bacteria, and variable regions of the sequence can be used for taxonomic identification.

Amplification bias

Uneven representation of regions of the DNA template in amplified DNA.

Bacterial and archaeal tree of life

The phylogenetic tree of all known bacteria and archaea based on the 16S ribosomal RNA gene.

Biofilm

A layered aggregate of microorganisms. These adherent cells are frequently embedded within a self-produced extracellular matrix that is generally composed of DNA, proteins and polysaccharides.

Candidate phyla

Uncultivated microbial groups that branch independently from known sequences near the base of the bacterial clade.

Chimaera

A recombinant molecule of DNA composed of segments from more than one source; multiple displacement amplification (MDA) can generate chimaeras that are predominantly inversions through its branching mechanism of DNA replication.

Endosymbiont

An organism that lives within the body or cells of another organism; it can include facultative or obligate symbionts.

Metagenomics

The study of the collective genomes contained in environmental samples using shotgun sequencing of DNA extracted from such samples.

Metatranscriptomic data

The set of all mRNA molecules or transcripts produced in a population of cells; they are typically obtained by shotgun sequencing of cDNA from a mixed microbial community.

Multiple displacement amplification

(MDA). A whole-genome DNA amplification method in which a DNA polymerase (usually the highly processive, strand-displacing Φ29 DNA polymerase) extends random primers while concurrently displacing the older products of downstream priming, which results in an exponential branching mechanism of DNA replication.

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Lasken, R., McLean, J. Recent advances in genomic DNA sequencing of microbial species from single cells. Nat Rev Genet 15, 577–584 (2014). https://doi.org/10.1038/nrg3785

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