A high-throughput assay for quantitative measurement of PCR errors

Dmitriy A Shagin,Dmitriy M Chudakov,Irina A Shagina,Andrew R Zaretsky,Mikhail Shugay,Ekaterina V Barsova,Sergey Lukyanov,Ilya V Kelmanson

doi:10.1038/s41598-017-02727-8

Abstract

The accuracy with which DNA polymerase can replicate a template DNA sequence is an extremely important property that can vary by an order of magnitude from one enzyme to another. The rate of nucleotide misincorporation is shaped by multiple factors, including PCR conditions and proofreading capabilities, and proper assessment of polymerase error rate is essential for a wide range of sensitive PCR-based assays. In this paper, we describe a method for studying polymerase errors with exceptional resolution, which combines unique molecular identifier tagging and high-throughput sequencing. Our protocol is less laborious than commonly-used methods, and is also scalable, robust and accurate. In a series of nine PCR assays, we have measured a range of polymerase accuracies that is in line with previous observations. However, we were also able to comprehensively describe individual errors introduced by each polymerase after either 20 PCR cycles or a linear amplification, revealing specific substitution preferences and the diversity of PCR error frequency profiles. We also demonstrate that the detected high-frequency PCR errors are highly recurrent and that the position in the template sequence and polymerase-specific substitution preferences are among the major factors influencing the observed PCR error rate.

Highlights

Polymerase error rate is a critical factor affecting the accuracy of a wide range of molecular biology techniques, including DNA cloning[1], PCR-based single-nucleotide polymorphism (SNP) and mutation detection[2] and library preparation for high-throughput sequencing[3, 4]
Using a DNA library that was not subjected to PCR amplification prior to sequencing, we demonstrate that the errors associated with high sequencing quality scores resemble the PCR error pattern, providing evidence for bridge-PCR amplification errors in high-filtered high-throughput sequencing data
We began by tagging each input template molecule with a random 14-mer nucleotide tag (UMI) in a linear amplification procedure, and performing PCR amplification with one of nine different assayed polymerases

Summary

Introduction

Polymerase error rate is a critical factor affecting the accuracy of a wide range of molecular biology techniques, including DNA cloning[1], PCR-based single-nucleotide polymorphism (SNP) and mutation detection[2] and library preparation for high-throughput sequencing[3, 4]. Sequencing errors, as the overall PCR error rate was estimated to be 0.06%10, while 454 sequencing can only reliably call variants with greater than 0.1% frequency[11] To overcome this limitation, we have turned to a technique based on unique molecular identifiers (UMI)[12,13,14], which makes it possible to trace individual DNA templates throughout different library preparation stages. We have turned to a technique based on unique molecular identifiers (UMI)[12,13,14], which makes it possible to trace individual DNA templates throughout different library preparation stages This technique has been successfully combined with high-throughput sequencing in various configurations for a wide range of applications that require precise quantification of rare variants[13, 15, 16]. Our analysis shows that the position in the template sequence and polymerase-specific substitution preferences are among the major factors influencing PCR error rate

Methods

Results

Conclusion