Effect of the enzyme and PCR conditions on the quality of high-throughput DNA sequencing results

Abstract

Library preparation protocols for high-throughput DNA sequencing (HTS) include amplification steps in which errors can build up. In order to have confidence in the sequencing data, it is important to understand the effects of different Taq polymerases and PCR amplification protocols on the DNA molecules sequenced. We compared thirteen enzymes in three different marker systems: simple, single copy nuclear gene and complex multi-gene family. We also tested a modified PCR protocol, which has been suggested to reduce errors associated with amplification steps. We find that enzyme choice has a large impact on the proportion of correct sequences recovered. The most complex marker systems yielded fewer correct reads, and the proportion of correct reads was greatly affected by the enzyme used. Modified cycling conditions did reduce the number of incorrect sequences obtained in some cases, but enzyme had a much greater impact on the number of correct reads. Thus, the coverage required for the safe identification of genotypes using one of the low quality enzymes could be seven times larger than with more efficient enzymes in a biallelic system with equal amplification of the two alleles. Consequently, enzyme selection for downstream HTS has important consequences, especially in complex genetic systems.

Optimization of high-throughput sequencing kinetics for determining enzymatic rate constants of thousands of RNA substrates

“…For instance, template concentration, bias against high GC templates, template switching, and polymerase errors may contribute to errors in downstream steps [27–29]. These previous studies indicated that these bias and errors can be minimized by using the minimum number of amplification cycles required to form products and defining the optimal template concentration in the PCR reaction.…”

Characterization of MHC class II B polymorphism in multiple populations of wild gorillas using non‐invasive samples and next‐generation sequencing

“…If introduced during early cycles of the PCR, these can potentially exceed the frequency of real alleles. The choice of the DNA polymerase and specifically its error rate has a profound impact on the quality of sequencing data [Brandariz‐Fontes et al, ]. Different enzymes vary in their processivity and specificity, especially in the presence of templates of inferior quality and/or with low‐copy number [Arezi et al, ; Moretti et al, ].…”

Methods for the extraction, storage, amplification and sequencing of DNA from environmental samples

“…It is well understood that different PCR conditions and different DNA polymerases will have different amplification biases and errors (Brandariz-Fontes et al 2015), which could impact the results of DNA metabarcoding studies. Ideally, a single enzyme and set of amplification conditions would be used across all DNA metabarcoding studies, but due to the large variation in sample media, primers, and taxa of interest, this is not possible.…”