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Rates and Mechanisms of Bacterial Mutagenesis from Maximum-Depth Sequencing

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.


(a) Barcodes are attached to original DNA molecules as per MDS protocol. After linear amplification, a second barcode is attached to the opposite end of each read (see Supplemental Information: Testing Sample Preparation and PCR Efficiency). Exponential PCR is then performed. In the analysis phase, reads can be grouped both by primary barcode (i.e. a classic MDS barcode family) and a second barcode corresponding to a “subfamily” of reads with the same parent from a particular linear amplification step before exponential amplification. (b) The probability that for a given family only reads of one subfamily are recovered (a “homogenous” barcode) decreases exponentially with R. For example, for R=3, the probability all 3 reads are of the same subfamily is 0.02. (c) We show the number of reads in each subfamily, sorted within each column by subfamily size, for the 1500 largest primary barcode families in the experiment. For families of such size, it is unlikely that a single subfamily will account for more than 25% of the total number of reads recovered from that family.
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Figure 6: (a) Barcodes are attached to original DNA molecules as per MDS protocol. After linear amplification, a second barcode is attached to the opposite end of each read (see Supplemental Information: Testing Sample Preparation and PCR Efficiency). Exponential PCR is then performed. In the analysis phase, reads can be grouped both by primary barcode (i.e. a classic MDS barcode family) and a second barcode corresponding to a “subfamily” of reads with the same parent from a particular linear amplification step before exponential amplification. (b) The probability that for a given family only reads of one subfamily are recovered (a “homogenous” barcode) decreases exponentially with R. For example, for R=3, the probability all 3 reads are of the same subfamily is 0.02. (c) We show the number of reads in each subfamily, sorted within each column by subfamily size, for the 1500 largest primary barcode families in the experiment. For families of such size, it is unlikely that a single subfamily will account for more than 25% of the total number of reads recovered from that family.

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) Barcodes are attached to original DNA molecules as per MDS protocol. After linear amplification, a second barcode is attached to the opposite end of each read (see Supplemental Information: Testing Sample Preparation and PCR Efficiency). Exponential PCR is then performed. In the analysis phase, reads can be grouped both by primary barcode (i.e. a classic MDS barcode family) and a second barcode corresponding to a “subfamily” of reads with the same parent from a particular linear amplification step before exponential amplification. (b) The probability that for a given family only reads of one subfamily are recovered (a “homogenous” barcode) decreases exponentially with R. For example, for R=3, the probability all 3 reads are of the same subfamily is 0.02. (c) We show the number of reads in each subfamily, sorted within each column by subfamily size, for the 1500 largest primary barcode families in the experiment. For families of such size, it is unlikely that a single subfamily will account for more than 25% of the total number of reads recovered from that family.
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Related In: Results  -  Collection

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Figure 6: (a) Barcodes are attached to original DNA molecules as per MDS protocol. After linear amplification, a second barcode is attached to the opposite end of each read (see Supplemental Information: Testing Sample Preparation and PCR Efficiency). Exponential PCR is then performed. In the analysis phase, reads can be grouped both by primary barcode (i.e. a classic MDS barcode family) and a second barcode corresponding to a “subfamily” of reads with the same parent from a particular linear amplification step before exponential amplification. (b) The probability that for a given family only reads of one subfamily are recovered (a “homogenous” barcode) decreases exponentially with R. For example, for R=3, the probability all 3 reads are of the same subfamily is 0.02. (c) We show the number of reads in each subfamily, sorted within each column by subfamily size, for the 1500 largest primary barcode families in the experiment. For families of such size, it is unlikely that a single subfamily will account for more than 25% of the total number of reads recovered from that family.
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.