﻿Comparing PCR-generated artifacts of different polymerases for improved accuracy of DNA metabarcoding

Satoshi Nagai,Satoshi Tazawa,Leila Basti,Sirje Sildever,Noriko Nishi,Takanori Kobayashi,Yoshizumi Ishino

doi:10.3897/mbmg.6.77704

Abstract

Accuracy of PCR amplification is vital for obtaining reliable amplicon-sequencing results by metabarcoding. Here, we performed a comparative analysis of error profiles in the PCR products by 14 different PCR kits using a mock eukaryotic community DNA sample mimicking metabarcoding analysis. To prepare a mock eukaryotic community from the marine environment, equal amounts of plasmid DNA from 40 microalgal species were mixed and used for amplicon-sequencing by a high-throughput sequencing approach. To compare the differences in PCR kits used for this experiment, we focused on the following seven parameters: 1) Quality, 2) Chimera, 3) Blast top hit accuracy, 4) Deletion, 5) Insertion, 6) Base substitution and 7) Amplification bias amongst species. The results showed statistically significant differences (p &lt; 0.05) for all of the seven parameters depending on the PCR kits used. These differences may result from the different DNA polymerases included in each kit, although the result can also be influenced by PCR reaction conditions. Simultaneous analysis of several parameters suggested that kits containing KOD plus Neo (TOYOBO) and HotStart Taq DNA polymerase (BiONEER, CA, US) at the annealing temperature of 65 °C displayed better results in terms of parameters associated with chimeras, top hit similarity and deletions.

Highlights

DNA polymerases are widely used for DNA manipulation in vitro, including DNA cloning, sequencing, labelling, mutagenesis and other purposes
All of the seven parameters used for the comparison of the sequence data from different PCR kits displayed statistically significant differences (p < 0.05; Table 3)
Significant differences amongst PCR kits were detected for all the parameters

Summary

Introduction

DNA polymerases are widely used for DNA manipulation in vitro, including DNA cloning, sequencing, labelling, mutagenesis and other purposes. The fundamental ability of DNA polymerases to synthesise a deoxyribonucleotide chain is conserved. The more specific properties, including thermostability, fidelity (proofreading activity), processivity (persistence of sequential nucleotide polymerisation) and specificity (proportion of non-specific amplification) vary depending on the polymerase. Thermostability can range from < 4 to 1380 min at 95 °C amongst DNA polymerases (van Pelt-Verkuil et al 2008). Some polymerases have a 3’-5’ proofreading activity that corrects occasionally-occurring nucleotide misincorporations during the extension processes. Some polymerases, for example, Taq DNA polymerase, lack proofreading ability, leaving the errors uncorrected (Taberlet et al 2018). For this reason, the usage of high-fidelity DNA polymerase has been suggested for reducing erroneous

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