Efficient de novo assembly of single-cell bacterial genomes from short-read data sets

Abstract

Whole genome amplification by the multiple displacement amplification (MDA) method allows sequencing of genomes from single cells of bacteria that cannot be cultured. However, genome assembly is challenging because of highly non-uniform read coverage generated by MDA. We describe an improved assembly approach tailored for single cell Illumina sequences that incorporates a progressively increasing coverage cutoff. This allows variable coverage datasets to be utilized effectively with assembly of E. coli and S. aureus single cell reads capturing >91% of genes within contigs, approaching the 95% captured from a multi-cell E. coli assembly. We apply this method to assemble a single cell genome of the uncultivated SAR324 clade of Deltaproteobacteria, a cosmopolitan bacterial lineage in the global ocean. Metabolic reconstruction suggests that SAR324 is aerobic, motile and chemotaxic. These new methods enable acquisition of genome assemblies for individual uncultivated bacteria, providing cell-specific genetic information absent from metagenomic studies.

Hadal biosphere: Insight into the microbial ecosystem in the deepest ocean on Earth

“…2). The abundance of SAR324, a potential chemolithoautotrophic deltaproteobacterial subgroup (6,23,24), also increased at 147 mbs and was detected as one of the predominant populations above 5000 mbs. In contrast, the potential heterotrophic SAR406 (Marine Group A) and Bacteroidetes (10, 25) dominated the prokaryotic SSU rRNA gene communities below 6,000 mbs, whereas they were found as only a minor population above 5,000 mbs.…”

Nearly finished genomes produced using gel microdroplet culturing reveal substantial intraspecies genomic diversity within the human microbiome

“…Single-cell amplifications were performed on the sorted single cells as previously described (Chitsaz et al 2011;Dupont et al 2012) with modifications being the 2-mL TE sorting volume and a total MDA reaction volume of 12.5 mL using the GenomiPhi HY Kit (GE Healthcare). Each frozen micromanipulated GMD was thawed on ice and placed in a thermocycler for 1 min at 65°C to melt the GMD and then briefly chilled on ice.…”

“…Incubation was performed on a thermocycler for 16 h at 30°C followed by a 10-min heat inactivation at 80°C and then held at 4°C. MDA reactions were diluted 20-fold, and subsequent PCR and 16S rDNA sequencing reactions were carried out as previously described (Chitsaz et al 2011;Dupont et al 2012).…”

“…Single cells were flow sorted at JCVI as described previously (Chitsaz et al 2011;Dupont et al 2012) with additional precautions taken to ensure safe flow sorting in the presence of potentially biohazardous microorganisms (Schmid et al 2003). Briefly, a FACS (fluorescence activated cell sorting) Aria II flow sorter (BD) equipped with a 70-mm nozzle, an argon ion laser providing 100 mW at 488 nm, and a custom forward scatter photomultiplier tube (FSC-PMT) for high-sensitivity detection of microorganisms was used for sorting of single cells.…”

“…GMD samples were stained with SYBR Green I and micromanipulated using techniques described previously (Chitsaz et al 2011;Dupont et al 2012). Briefly, we used an Olympus IX70 inverted microscope equipped with a TransferMan NK2 and CellTram Vario (Eppendorf) with sterile glass capillaries blunted to an I.D.…”