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jueves, 23 de junio de 2016

Rates and mechanisms of bacterial mutagenesis from maximum-depth sequencing

In 1943, Luria and Delbrück used a phage-resistance assay to establish spontaneous mutation as a driving force of microbial diversity1. Mutation rates are still studied using such assays, but these can only be used to examine the small minority of mutations conferring survival in a particular condition. Newer approaches, such as long-term evolution followed by whole-genome sequencing23, may be skewed by mutational ‘hot’ or ‘cold’ spots34. Both approaches are affected by numerous caveats567. Here we devise a method, maximum-depth sequencing (MDS), to detect extremely rare variants in a population of cells through error-corrected, high-throughput sequencing. We directly measure locus-specific mutation rates in Escherichia coli and show that they vary across the genome by at least an order of magnitude. Our data suggest that certain types of nucleotide misincorporation occur 104-fold more frequently than the basal rate of mutations, but are repaired in vivo. Our data also suggest specific mechanisms of antibiotic-induced mutagenesis, including downregulation of mismatch repair via oxidative stress, transcription–replication conflicts, and, in the case of fluoroquinolones, direct damage to DNA.

De novo mutations in bacteria remain a notoriously difficult target for high-throughput sequencing. Whereas E. coli mutate fewer than 1 in 109 bases per generation, high-fidelity polymerases used for library preparation polymerase chain reaction (PCR) cause errors in ~4 out of 106 bases8. Illumina machines misread ~1 in 103 bases9. Recent methods, such as barcoding of reads from the same original DNA molecule8, have lowered the error rate of sequencing. However, such methods can have low yields10 and do not address errors introduced by PCR. PCR errors can be overcome using duplex barcoding, which forms a consensus from both strands of a DNA template molecule11. However, even when a small region is targeted12, duplexing lowers yield even further. The mutational landscape of an RNA virus with mutation rate 104-fold greater than E. coli was recently mapped using ‘circle sequencing’. However, this technique is not designed for targeted coverage of a single locus, and its accuracy is limited by sequence read length1013.