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European Team Uses Illumina Sequencing to Assess E. Coli Mutation Patterns

NEW YORK (GenomeWeb News) – A new paper suggests high-throughput sequencing can offer a window into how mutations occur in bacterial genomes.

German and French researchers used an Illumina platform to sequence the genomes of several Escherichia coli colonies that had been chemically mutagenized, comparing them with the original, non-mutated strain. The research, which appeared online last night in the Proceedings of the National Academy of Sciences, suggests mutations in the E. coli genome don't happen randomly but rather in patterns that offer clues about the cellular processes involved.

"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," senior author Vasily Ogryzko, a researcher at the University of Paris-Sud, and his co-authors wrote.

Although past studies have provided estimates of the mutation rate of E. coli and other bugs, the team noted, a precise understanding of mutations and genetic variability has been difficult to ascertain.

With the advent of high throughput sequencing and other genomic approaches, they added, it's possible to look at how mutation and recombination occurs across the genomes of single cells — an idea they tested using a well studied E. coli strain.

"Recent advances in high throughput genomic analysis open up new opportunities for analysis of genome variability," the researchers wrote. "[W]e set out to explore how this analysis can help in observing, at the single-cell level, the contributions and interactions between the molecular processes contributing to mutation generation and segregation."

To do this, the team first mutagenized a strain of E. coli called K-12 CC102 using the chemical ethyl methanesulfonate, or EMS. They then selected bacterial colonies at random, isolated genomic DNA, and used an Illumina Genome Analyzer to sequence the mutagenized genomes.

Based on their comparison of the unmutagenized CC102 strain and six mutagenized colonies, the team found 70 mutations per genome, on average. Most of these mutations involved G:C nucleotides being converted to A:T nucleotides — consistent with the known action of the EMS chemical.

In general, the researchers noted, the mutations tended to occur in specific patterns. For instance, the researchers found that mutations clustered in different parts of the genome — which they speculated may be caused by factors such as recombination, DNA synthesis, and DNA repair.

The team also found stretches in which only G to A changes occurred followed by other regions with just C to T changes. Their subsequent experiments indicated that homologous recombination might contribute to the switching between these mutations.

Even so, the team explained, more research, including additional sequencing work, is needed to better understand the processes behind some of the other mutations detected.

"Whereas most of the observed distribution patterns can be explained by the known features of enzymatic processes (semi-conservative DNA replication, homologous recombination, competition between replication and repair), the source of others remains to be elucidated," they wrote.