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Modular tagging of amplicons using a single PCR for high-throughput sequencing


Clarke, LJ and Czechowski, P and Soubrier, J and Stevens, MI and Cooper, A, Modular tagging of amplicons using a single PCR for high-throughput sequencing, Molecular Ecology Resources, 14, (1) pp. 117-121. ISSN 1755-098X (2014) [Refereed Article]

Copyright Statement

Copyright 2013 John Wiley & Sons Ltd

DOI: doi:10.1111/1755-0998.12162


High-throughput sequencing (HTS) of PCR amplicons is becoming the method of choice to sequence one or several targeted loci for phylogenetic and DNA barcoding studies. Although the development of HTS has allowed rapid generation of massive amounts of DNA sequence data, preparing amplicons for HTS remains a rate-limiting step. For example, HTS platforms require platform-specific adapter sequences to be present at the 5′ and 3′ end of the DNA fragment to be sequenced. In addition, short multiplex identifier (MID) tags are typically added to allow multiple samples to be pooled in a single HTS run. Existing methods to incorporate HTS adapters and MID tags into PCR amplicons are either inefficient, requiring multiple enzymatic reactions and clean-up steps, or costly when applied to multiple samples or loci (fusion primers). We describe a method to amplify a target locus and add HTS adapters and MID tags via a linker sequence using a single PCR. We demonstrate our approach by generating reference sequence data for two mitochondrial loci (COI and 16S) for a diverse suite of insect taxa. Our approach provides a flexible, cost-effective and efficient method to prepare amplicons for HTS.

Item Details

Item Type:Refereed Article
Keywords:16S large ribosomal subunit, amplicon sequencing, cytochrome c oxidase subunit I, DNA barcoding, high-throughput sequencing, insect, MoTASP, multiplex identifier
Research Division:Biological Sciences
Research Group:Genetics
Research Field:Genetics not elsewhere classified
Objective Division:Expanding Knowledge
Objective Group:Expanding knowledge
Objective Field:Expanding knowledge in the biological sciences
UTAS Author:Clarke, LJ (Dr Laurence Clarke)
ID Code:104023
Year Published:2014
Web of Science® Times Cited:25
Deposited By:CRC-Antarctic Climate & Ecosystems
Deposited On:2015-11-01
Last Modified:2017-11-01

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