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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). NA. ISSN NA

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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
    Date Deposited: 28 Jan 2015 10:48
    Last Modified: 29 Jan 2015 08:48
    URI: http://repository.usp.ac.fj/id/eprint/7885
    UNSPECIFIED

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