Use of high throughput sequencing to observe genome dynamics at a single cell level

Abstract

With the development of high throughput sequencing technology, it becomes possible to directly analyze mutation distribution in a genome-wide fashion, dissociating mutation rate measurements from the traditional underlying assumptions. Here, we sequenced several genomes of Escherichia coli from colonies obtained after chemical mutagenesis and observed a strikingly nonrandom distribution of the induced mutations. These include long stretches of exclusively G to A or C to T transitions along the genome and orders of magnitude intra-and intergenomic differences in mutation density. Whereas most of these observations can be explained by the known features of enzymatic processes, the others could reflect stochasticity in the molecular processes at the single-cell level. Our results demonstrate how analysis of the molecular records left in the genomes of the descendants of an individual mutagenized cell allows for genome-scale observations of fixation and segregation of mutations, as well as recombination events, in the single genome of their progenitor.mutation ͉ stochasticity ͉ semiconservative ͉ recombination ͉ genome-wide

Clusters of Multiple Mutations: Incidence and Molecular Mechanisms

“…Thus, if lesions are nucleotide- or motif-specific, mutations in the cluster would be strand-coordinated, i.e., mutations of the same kind would be found in the same strand. These phenomena were documented in several whole-genome sequenced (WGS) clones of E. coli grown from cells treated acutely with ethyl methanesulfonate (EMS), a mutagen which is highly specific for inducing G→A changes (88). Cluster sizes were in the hundreds of kb, and mutations were either nearly all G→A or nearly all C→T (as expected if G→A changes occurred in the opposite strand).…”

RNA-seq based transcriptomic analysis of single bacterial cells

“…3,4,5,7,8,9,11,12,13,14,15,17,18) * CV is equal to standard deviation/average from cell-1,2,3 and cell-4,5,6 for 24 h and 72 h, respectively, "1" indicated that we used the data of 424 transcripts detected in all single cells; "2" indicated that we used the data of 3117 transcripts detected in at least one single cell. 3,4,5,7,8,9,11,12,13,14,15,17,18) * CV is equal to standard deviation/average from cell-1,2,3 and cell-4,5,6 for 24 h and 72 h, respectively, "1" indicated that we used the data of 424 transcripts detected in all single cells; "2" indicated that we used the data of 3117 transcripts detected in at least one single cell.…”

Two Mechanisms Produce Mutation Hotspots at DNA Breaks in Escherichia coli

“…Spontaneous mutations in mice fall in ∼30 kb showers of simultaneous multiple mutations (Drake, 2007b; Wang et al., 2007). Both chemically mutagenized yeast (Burch et al., 2011) and E. coli (Parkhomchuk et al., 2009) show local clusters of mutations, as do the genomes of human breast (Nik-Zainal et al., 2012) and colon (Roberts et al., 2012) cancer cells, and chemically treated yeast (Ma et al., 2012). These and other observations (Caporale, 2006; Drake, 2007a, 2007b) indicate that the processes of mutagenesis themselves, and not just the sites in which mutations are tolerated, can be localized in genomes and are not distributed randomly.…”