Premium
Modular tagging of amplicons using a single PCR for high‐throughput sequencing
Author(s) -
Clarke Laurence J.,
Czechowski Paul,
Soubrier Julien,
Stevens Mark I.,
Cooper Alan
Publication year - 2014
Publication title -
molecular ecology resources
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.96
H-Index - 136
eISSN - 1755-0998
pISSN - 1755-098X
DOI - 10.1111/1755-0998.12162
Subject(s) - amplicon , biology , adapter (computing) , computational biology , multiplex , crispr , dna sequencing , genetics , polymerase chain reaction , dna , computer science , gene , computer hardware
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 16 S ) for a diverse suite of insect taxa. Our approach provides a flexible, cost‐effective and efficient method to prepare amplicons for HTS .