High throughput sequencing of multiple amplicons for barcoding and integrative taxonomy

Until now, the potential of NGS has been seldom realised for the construction of barcode reference libraries. Using a two-step PCR approach and MiSeq sequencing, we tested a cost-effective method and developed a custom workflow to simultaneously sequence multiple markers (COI, Cytb and EF, altogether 2kb) from hundreds of specimens. Interestingly, primers and PCR conditions used for Sanger sequencing did not require optimisation to construct MiSeq library. After completion of quality controls, 87% of the species and 76% of the specimens had valid sequences for the three markers. Nine specimens (3%) exhibited two divergent (up to 10%) sequence clusters. In 95% of the species, MiSeq and Sanger