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Type: Journal article
Title: Modular tagging of amplicons using a single PCR for high-throughput sequencing
Author: Clarke, L.
Czechowski, P.
Soubrier, J.
Stevens, M.
Cooper, A.
Citation: Molecular Ecology Resources, 2014; 14(1):117-121
Publisher: Wiley
Issue Date: 2014
ISSN: 1755-098X
Statement of
Laurence J. Clarke, Paul Czechowski, Julien Soubrier, Mark I. Stevens and Alan Cooper
Abstract: 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.
Keywords: 16S large ribosomal subunit; amplicon sequencing; cytochrome c oxidase subunit I; DNA barcoding; high-throughput sequencing; insect; MoTASP; multiplex identifier
Rights: © 2013 John Wiley & Sons Ltd
DOI: 10.1111/1755-0998.12162
Grant ID: ARC
Appears in Collections:Aurora harvest 2
Earth and Environmental Sciences publications

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