Polymerase specific error rates and profiles identified by single molecule sequencing

Abstract

DNA polymerases have an innate error rate which is polymerase and DNA context specific. Historically the mutational rate and profiles have been measured using a variety of methods, each with their own technical limitations. Here we used the unique properties of single molecule sequencing to evaluate the mutational rate and profiles of six DNA polymerases at the sequence level. In addition to accurately determining mutations in double strands, single molecule sequencing also captures direction specific transversions and transitions through the analysis of heteroduplexes. Not only did the error rates vary, but also the direction specific transitions differed among polymerases.

Examining Sources of Error in PCR by Single-Molecule Sequencing

“…Hestand et al . measured the fidelity of Taq polymerase to be 4.4 × 10 −5 errors/base/cycle, and when normalized for the number of PCR cycles, our Taq measurements of 5.7 × 10 −5 sub/base/cycle are similar [15]. Phusion DNA polymerase (1.3 × 10 −6 sub/base/cycle in this study) was found to have a similar error rate by single-molecule sequencing (1.8 × 10 −6 sub/base/cycle) [15], but a lower error rate by another Illumina-based method (4.5 × 10 −7 total errors/base/cycle) [5], indicating there may be some discrepancy in the literature regarding the error rate of Phusion DNA polymerase.…”

“…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.…”

“…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]. …”

“…Using PacBio SMRT sequencing, Hestand et al . measured the base substitution error rates of several PCR enzymes [15]. Single-molecule sequencing of individual amplification products by next-generation sequencing has made it easier to assay polymerase fidelity, but previous studies were limited to examining polymerase base substitution errors.…”