22 research outputs found
Single-cell enabled comparative genomics of a deep ocean SAR11 bathytype.
This is the author's accepted mansucript.Final version available from Nature via the DOI in this record.Bacterioplankton of the SAR11 clade are the most abundant microorganisms in marine systems, usually representing 25% or more of the total bacterial cells in seawater worldwide. SAR11 is divided into subclades with distinct spatiotemporal distributions (ecotypes), some of which appear to be specific to deep water. Here we examine the genomic basis for deep ocean distribution of one SAR11 bathytype (depth-specific ecotype), subclade Ic. Four single-cell Ic genomes, with estimated completeness of 55%-86%, were isolated from 770 m at station ALOHA and compared with eight SAR11 surface genomes and metagenomic datasets. Subclade Ic genomes dominated metagenomic fragment recruitment below the euphotic zone. They had similar COG distributions, high local synteny and shared a large number (69%) of orthologous clusters with SAR11 surface genomes, yet were distinct at the 16S rRNA gene and amino-acid level, and formed a separate, monophyletic group in phylogenetic trees. Subclade Ic genomes were enriched in genes associated with membrane/cell wall/envelope biosynthesis and showed evidence of unique phage defenses. The majority of subclade Ic-specfic genes were hypothetical, and some were highly abundant in deep ocean metagenomic data, potentially masking mechanisms for niche differentiation. However, the evidence suggests these organisms have a similar metabolism to their surface counterparts, and that subclade Ic adaptations to the deep ocean do not involve large variations in gene content, but rather more subtle differences previously observed deep ocean genomic data, like preferential amino-acid substitutions, larger coding regions among SAR11 clade orthologs, larger intergenic regions and larger estimated average genome size.This work was supported by the Gordon and Betty Moore Foundation (SJG and EFD), the US Department of Energy Joint Genome Institute (JGI) Community Supported Program grant 2011-387 (RS, BKS, EFD, SJG), National Science Foundation (NSF) Science and Technology Center Award EF0424599 (EFD), NSF awards EF-826924 (RS), OCE-821374 (RS) and OCE-1232982 (RS and BKS), and is based on work supported by the NSF under Award no. DBI-1003269 (JCT). Sequencing was conducted by JGI and supported by the Office of Science of the US Department of Energy under Contract No. DE-AC02-05CH11231
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
Dissecting genomic diversity, one cell at a time
Emerging technologies are bringing single-cell genome sequencing into the mainstream; this field has already yielded insights into the genetic architecture and variability between cells that highlight the dynamic nature of the genome
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