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dc.contributor.authorLawrence, Dylan
dc.contributor.authorCampbell, Danielle E
dc.contributor.authorSchriefer, Lawrence A
dc.contributor.authorRodgers, Rachel
dc.contributor.authorWalker, Forrest C
dc.contributor.authorTurkin, Marissa
dc.contributor.authorDroit, Lindsay
dc.contributor.authorParkes, Miles
dc.contributor.authorHandley, Scott A
dc.contributor.authorBaldridge, Megan T
dc.description.abstractAs our understanding of the importance of the human microbiota in health and disease grows, so does our need to carefully resolve and delineate its genomic content. 16S rRNA gene-based analyses yield important insights into taxonomic composition, and metagenomics-based approaches reveal the functional potential of microbial communities. However, these methods generally fail to directly link genetic features, including bacterial genes and mobile genetic elements, to each other and to their source bacterial genomes. Further, they are inadequate to capture the microdiversity present within a genus, species, or strain of bacteria within these complex communities. Here, we present a method utilizing fluorescence-activated cell sorting for isolation of single bacterial cells, amplifying their genomes, screening them by 16S rRNA gene analysis, and selecting cells for genomic sequencing. We apply this method to both a cultured laboratory strain of Escherichia coli and human stool samples. Our analyses reveal the capacity of this method to provide nearly complete coverage of bacterial genomes when applied to isolates and partial genomes of bacterial species recovered from complex communities. Additionally, this method permits exploration and comparison of conserved and variable genomic features between individual cells. We generate assemblies of novel genomes within the Ruminococcaceae family and the Holdemanella genus by combining several 16S rRNA gene-matched single cells, and report novel prophages and conjugative transposons for both Bifidobacterium and Ruminococcaceae. Thus, we demonstrate an approach for flow cytometric separation and sequencing of single bacterial cells from the human microbiota, which yields a variety of critical insights into both the functional potential of individual microbes and the variation among those microbes. This method definitively links a variety of conserved and mobile genomic features, and can be extended to further resolve diverse elements present in the human microbiota.
dc.publisherInforma UK Limited
dc.rightsAttribution 4.0 International
dc.sourcenlmid: 101495343
dc.sourceessn: 1949-0984
dc.subjectSingle cell
dc.subjectMobile genetic elements
dc.subjectGenomic Assembly
dc.subjectSingle-amplified Genome
dc.subjectFlow Cytometry
dc.subjectInterspersed Repetitive Sequences
dc.subjectGenome, Bacterial
dc.subjectSingle-Cell Analysis
dc.subjectHigh-Throughput Nucleotide Sequencing
dc.subjectGastrointestinal Microbiome
dc.titleSingle-cell genomics for resolution of conserved bacterial genes and mobile genetic elements of the human intestinal microbiota using flow cytometry.
prism.publicationNameGut Microbes
dc.contributor.orcidBaldridge, Megan T [0000-0002-7030-6131]
pubs.funder-project-idNHGRI NIH HHS (T32 HG000045)
pubs.funder-project-idNIH HHS (R01 OD024917)
pubs.funder-project-idNIDDK NIH HHS (RC2 DK116713, T32 DK077653)

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Attribution 4.0 International
Except where otherwise noted, this item's licence is described as Attribution 4.0 International