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

Brandariz-Fontes, Claudia and Camacho-Sanchez, Miguel and Vila`, Carles and Vega-Pla, Jose´ Luis and Rico, Ciro and Leonard, Jennifer A. (2015) Effect of the enzyme and PCR conditions on the quality of high-throughput DNA sequencing results. Scientific Reports, 5 (8056). pp. 1-5. ISSN 2045-2322

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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 DNAmolecules 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.

Item Type:	Journal Article
Additional Information:	DOI:10.1038/srep08056
Subjects:	Q Science > Q Science (General) Q Science > QH Natural history > QH301 Biology Q Science > QH Natural history > QH426 Genetics
Divisions:	Faculty of Science, Technology and Environment (FSTE) > School of Marine Studies
Depositing User:	Fulori Nainoca - Waqairagata
Date Deposited:	27 Jan 2015 22:48
Last Modified:	02 Oct 2018 03:41
URI:	https://repository.usp.ac.fj/id/eprint/7885

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