Abstract
BackgroundSingle cell genomics (SCG) is a combination of methods whose goal is to decipher the complete genomic sequence from a single cell and has been applied mostly to organisms with smaller genomes, such as bacteria and archaea. Prior single cell studies showed that a significant portion of a genome could be obtained. However, breakages of genomic DNA and amplification bias have made it very challenging to acquire a complete genome with single cells. We investigated an artificial method to induce polyploidy in Bacillus subtilis ATCC 6633 by blocking cell division and have shown that we can significantly improve the performance of genomic sequencing from a single cell.Methodology/Principal FindingsWe inhibited the bacterial cytoskeleton protein FtsZ in B. subtilis with an FtsZ-inhibiting compound, PC190723, resulting in larger undivided single cells with multiple copies of its genome. qPCR assays of these larger, sorted cells showed higher DNA content, have less amplification bias, and greater genomic recovery than untreated cells.SignificanceThe method presented here shows the potential to obtain a nearly complete genome sequence from a single bacterial cell. With millions of uncultured bacterial species in nature, this method holds tremendous promise to provide insight into the genomic novelty of yet-to-be discovered species, and given the temporary effects of artificial polyploidy coupled with the ability to sort and distinguish differences in cell size and genomic DNA content, may allow recovery of specific organisms in addition to their genomes.
Highlights
Microbial communities are complex ’supra-organisms’ consisting of bacteria, archaea, eukaryotes, and viruses that interact with and depend upon one another in order to survive and thrive in their natural environmental niches
B. subtilis Responded to Treatment with PC190723 Resulting in Larger Cells
Cytographic data from flow cytometry showed that PC190723-treated cells became larger, as measured by both side and forward scatter, during the course of treatment compared to untreated control cells (Fig. 1A)
Summary
Microbial communities are complex ’supra-organisms’ consisting of bacteria, archaea, eukaryotes, and viruses that interact with and depend upon one another in order to survive and thrive in their natural environmental niches. Whole genome sequencing with amplified DNA has been tested on single cells of several target species such as E. coli and Prochlorococcus [9], and has been used for novel de novo sequencing of TM7 [10,11], flavobacteria [12,13], and Prochlorococcus single cells [14]. These published efforts resulted in fragmented genome assemblies with up to a thousand contigs or more, extensive and expensive genome closure efforts can reduce this to a much smaller number [13]. We investigated an artificial method to induce polyploidy in Bacillus subtilis ATCC 6633 by blocking cell division and have shown that we can significantly improve the performance of genomic sequencing from a single cell
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