Department of Energy Joint Genome Institute, Los Alamos National Laboratory, Los Alamos, New Mexico, United States of America.
PLoS One. 2012;7(5):e37387. doi: 10.1371/journal.pone.0037387. Epub 2012 May 22.
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.
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.
单细胞基因组学（SCG）是多种方法的组合，其目标是从单个细胞中破译完整的基因组序列，并且主要应用于基因组较小的生物体，如细菌和古菌。先前的单细胞研究表明，可以获得基因组的很大一部分。然而，基因组 DNA 的断裂和扩增偏差使得从单个细胞中获得完整基因组变得极具挑战性。我们研究了一种通过阻断细胞分裂在枯草芽孢杆菌 ATCC 6633 中诱导多倍体的人工方法，结果表明我们可以显著提高从单个细胞进行基因组测序的性能。
方法/主要发现：我们用一种抑制 FtsZ 的化合物 PC190723 抑制枯草芽孢杆菌中的细菌细胞骨架蛋白 FtsZ，导致其基因组出现多个重复的、未分裂的大单细胞。这些较大的、经分选的细胞的 qPCR 检测显示，其 DNA 含量更高，扩增偏差更小，基因组回收率更高，与未经处理的细胞相比。
这里提出的方法显示了从单个细菌细胞中获得几乎完整的基因组序列的潜力。自然界中有数以百万计的未培养细菌物种，这种方法为了解尚未发现的物种的基因组新颖性提供了巨大的潜力，并且考虑到人工多倍体的暂时影响以及分选和区分细胞大小和基因组 DNA 含量差异的能力，除了它们的基因组之外，还可能恢复特定的生物体。
