Mapping DNA polymerase errors by single-molecule sequencing

Abstract

Genomic integrity is compromised by DNA polymerase replication errors, which occur in a sequence-dependent manner across the genome. Accurate and complete quantification of a DNA polymerase's error spectrum is challenging because errors are rare and difficult to detect. We report a high-throughput sequencing assay to map in vitro DNA replication errors at the single-molecule level. Unlike previous methods, our assay is able to rapidly detect a large number of polymerase errors at base resolution over any template substrate without quantification bias. To overcome the high error rate of high-throughput sequencing, our assay uses a barcoding strategy in which each replication product is tagged with a unique nucleotide sequence before amplification. This allows multiple sequencing reads of the same product to be compared so that sequencing errors can be found and removed. We demonstrate the ability of our assay to characterize the average error rate, error hotspots and lesion bypass fidelity of several DNA polymerases.

Examining Sources of Error in PCR by Single-Molecule Sequencing

“…For example, Taq polymerase (1.8 × 10 −4 total errors/base/doubling) was found to have a similar error rate as measured by a primer extension assay (2.0 × 10 −4 errors/base) [16]. The base substitution rate of Q5 DNA polymerase (5.3 × 10 −7 sub/base/doubling, in this study) was also measured by a primer extension assay to be 4.0 × 10 −6 substitutions/base, higher than what was measured in this study, although this number could possibly include errors introduced by DNA damage as well as polymerase errors [16]. In the primer extension fidelity assay by Lee et al ., one extension product from Q5 polymerase contained abnormally high C→A substitutions and was excluded from analysis [16].…”

“…DNA polymerase replication fidelity has been extensively studied with multiple methods and various assay conditions. Assays to determine replication fidelity have utilized various approaches: blue/white screening ([7], [8], [9], and reviewed in [10]), forward mutation [11], denaturing gradient gel electrophoresis [12, 13], high throughput Sanger sequencing [14], or next-generation sequencing [5, 15, 16]. Differences in assay methodology, reaction conditions, template sequences and error reporting units can yield different absolute values for error rates.…”

“…Normalizing raw error rates to the number of template doublings corrects for the propagation of errors during exponential amplification and the different replication efficiencies of different polymerases. However, more recent fidelity studies utilizing next-generation sequencing typically report error rates as error per base per number of PCR cycles [5, 15, 16]. As DNA replication per PCR cycle is not perfectly efficient, the number of doubling events is less than the number of PCR cycles, making it more difficult to compare studies with different error reporting units.…”

“…Lee et al . also used a similar approach to measure the fidelity of DNA polymerases performing a single round of DNA synthesis [16]. In both methods, individual molecules were tagged with a unique molecular index prior to Illumina sequencing.…”

“…Further studies indicated that the background error rate of this method is approximately 2.5 × 10 −6 errors/base based on the integrity of the molecular index [3, 17]. The error rates of very accurate polymerases also fall in this range, thus highlighting the challenge of measuring their fidelities [5, 15, 16]. …”