Artificial Polyploidy Improves Bacterial Single Cell Genome Recovery

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.

Comparative Metagenomics of Viral Assemblages Inhabiting Four Phyla of Marine Invertebrates

“…These results reflect the fact that the phi29 DNA polymerase preferentially amplifies genomic regions associated with circular ssDNA viruses during initial priming events (Dichosa et al, 2012). We agree with previous work and suggest that researchers choose an amplification strategy that will minimally bias results if viral community proportions are to be studied.…”

Single cell genomics: an individual look at microbes

“…For example, environmental sample preservation has a significant impact on cell lysis efficiency, which may impact WGA evenness and genome assembly quality (unpublished data). Natural [30 ] and artificially induced [57] polyploidy can improve single cell genome Single cell genomics Stepanauskas 617…”

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

“…It is therefore generally not possible to finish a microbial genome starting from a single cell (Rodrigue et al 2009). Because the bias is lessened with an additional starting template, a potential solution is to provide more copies of the target genome as input into an MDA (Pan et al 2008;Woyke et al 2010;Dichosa et al 2012). Here we propose using bacterial microcolonies contained within gel microdroplets (GMDs), which allows one to perform DNA amplification on hundreds to thousands of copies of a bacterial genome.…”