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Rates and Mechanisms of Bacterial Mutagenesis from Maximum-Depth Sequencing

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ABSTRACT

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 examine the small minority of mutations conferring survival in a particular condition. Newer approaches, such as long-term evolution followed by whole-genome sequencing 2, 3, may be skewed by mutational “hot” or “cold” spots 3, 4. Both approaches are affected by numerous caveats 5, 6, 7 (see Supplemental Information). 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 E. 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.

No MeSH data available.


(a) Mock culture composed of rpoB point mutants of known concentration was sequenced using MDS. Output concentrations of each point mutant recovered from R=2 analysis are plotted against its input concentration (see Supplemental Table 2 for details). (b-c) Distribution of the sizes of barcode families in four trials, shown as log10(# barcode families) per trial vs. size of barcode family in reads (R). (b) Trials used for the calibration run shown in (a) (~100M reads total, divided into four trials) (c) Representative quadruplicate trials (from rpoB of WT bacteria grown in LB broth with no antibiotics) taking up a total of one quarter of the output of a HiSeq rapid run, a total of ~60M reads.
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Figure 5: (a) Mock culture composed of rpoB point mutants of known concentration was sequenced using MDS. Output concentrations of each point mutant recovered from R=2 analysis are plotted against its input concentration (see Supplemental Table 2 for details). (b-c) Distribution of the sizes of barcode families in four trials, shown as log10(# barcode families) per trial vs. size of barcode family in reads (R). (b) Trials used for the calibration run shown in (a) (~100M reads total, divided into four trials) (c) Representative quadruplicate trials (from rpoB of WT bacteria grown in LB broth with no antibiotics) taking up a total of one quarter of the output of a HiSeq rapid run, a total of ~60M reads.

Mentions: We introduce Maximum-Depth Sequencing (MDS) for detecting extremely rare variants in any region of interest (ROI) in a population of cells (See Methods, Fig. 1a). By synthesizing unique barcodes directly onto the ROI of an original genomic DNA molecule and then copying that molecule using linear amplification, we increase yield (Fig. 1B) and drown out both polymerase and sequencing errors (Fig. 1C). On mock cultures with single-nucleotide mutants spiked in at known concentrations, MDS reliably recovers the expected proportion of mutants at the lowest frequency tested, 10−6 (Extended Data Fig. 1). On in vitro synthesized DNA templates, MDS reduces the error rate to less than 5×10−8 per nucleotide sequenced (Fig. 1C, Extended Data Fig. 2). By increasing the number of reads used to call a consensus sequence (R), MDS can lower error rate indefinitely, given sufficient coverage (Methods: Error Rate of MDS). Application of a second barcode after linear PCR increases accuracy at an even sharper rate and was used here to demonstrate library preparation efficiency (Extended Data Fig. 2 and Supplemental Information: Testing Sample Preparation and PCR Efficiency.)


Rates and Mechanisms of Bacterial Mutagenesis from Maximum-Depth Sequencing
(a) Mock culture composed of rpoB point mutants of known concentration was sequenced using MDS. Output concentrations of each point mutant recovered from R=2 analysis are plotted against its input concentration (see Supplemental Table 2 for details). (b-c) Distribution of the sizes of barcode families in four trials, shown as log10(# barcode families) per trial vs. size of barcode family in reads (R). (b) Trials used for the calibration run shown in (a) (~100M reads total, divided into four trials) (c) Representative quadruplicate trials (from rpoB of WT bacteria grown in LB broth with no antibiotics) taking up a total of one quarter of the output of a HiSeq rapid run, a total of ~60M reads.
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Related In: Results  -  Collection

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Figure 5: (a) Mock culture composed of rpoB point mutants of known concentration was sequenced using MDS. Output concentrations of each point mutant recovered from R=2 analysis are plotted against its input concentration (see Supplemental Table 2 for details). (b-c) Distribution of the sizes of barcode families in four trials, shown as log10(# barcode families) per trial vs. size of barcode family in reads (R). (b) Trials used for the calibration run shown in (a) (~100M reads total, divided into four trials) (c) Representative quadruplicate trials (from rpoB of WT bacteria grown in LB broth with no antibiotics) taking up a total of one quarter of the output of a HiSeq rapid run, a total of ~60M reads.
Mentions: We introduce Maximum-Depth Sequencing (MDS) for detecting extremely rare variants in any region of interest (ROI) in a population of cells (See Methods, Fig. 1a). By synthesizing unique barcodes directly onto the ROI of an original genomic DNA molecule and then copying that molecule using linear amplification, we increase yield (Fig. 1B) and drown out both polymerase and sequencing errors (Fig. 1C). On mock cultures with single-nucleotide mutants spiked in at known concentrations, MDS reliably recovers the expected proportion of mutants at the lowest frequency tested, 10−6 (Extended Data Fig. 1). On in vitro synthesized DNA templates, MDS reduces the error rate to less than 5×10−8 per nucleotide sequenced (Fig. 1C, Extended Data Fig. 2). By increasing the number of reads used to call a consensus sequence (R), MDS can lower error rate indefinitely, given sufficient coverage (Methods: Error Rate of MDS). Application of a second barcode after linear PCR increases accuracy at an even sharper rate and was used here to demonstrate library preparation efficiency (Extended Data Fig. 2 and Supplemental Information: Testing Sample Preparation and PCR Efficiency.)

View Article: PubMed Central - PubMed

ABSTRACT

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 examine the small minority of mutations conferring survival in a particular condition. Newer approaches, such as long-term evolution followed by whole-genome sequencing 2, 3, may be skewed by mutational “hot” or “cold” spots 3, 4. Both approaches are affected by numerous caveats 5, 6, 7 (see Supplemental Information). 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 E. 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.

No MeSH data available.