Artificial Polyploidy Improves Bacterial Single Cell Genome Recovery

BACKGROUND: Single 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 FINDINGS: We 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. SIGNIFICANCE: The 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

Dimpled disks and baskets

Speleothem: FMD, Sample color: Chocolate Brownhttps://digitalrepository.unm.edu/kc_fmd/1392/thumbnail.jp

Overview of tissue-paper chunk with filametns

Speleothem: FMD, Sample color: Steel Grayhttps://digitalrepository.unm.edu/kc_fmd/1389/thumbnail.jp

Bridging filaments across tissue-paper

Speleothem: FMD, Sample color: Steel Grayhttps://digitalrepository.unm.edu/kc_fmd/1431/thumbnail.jp

Overview of dimple disk aggregate

Speleothem: FMD, Sample color: Chocolate Brownhttps://digitalrepository.unm.edu/kc_fmd/1433/thumbnail.jp

Intertwining structure filament 1

Speleothem: FMD, Sample color: Steel Grayhttps://digitalrepository.unm.edu/kc_fmd/1391/thumbnail.jp

Close up of spheroids and segmented hairy filaments

Speleothem: FMD, Sample color: Steel Grayhttps://digitalrepository.unm.edu/kc_fmd/1408/thumbnail.jp

Corroded cuboid crystals

https://digitalrepository.unm.edu/kc_fmd/1421/thumbnail.jp

EDX of dimpled disks from 03118b48.tif

Speleothem: FMD, Sample color: Chocolate Brownhttps://digitalrepository.unm.edu/kc_fmd/1447/thumbnail.jp

EDX of dimpled disks from 03118b40.tif

Speleothem: FMD, Sample color: Chocolate Brownhttps://digitalrepository.unm.edu/kc_fmd/1405/thumbnail.jp