PT  - JOURNAL ARTICLE
AU  - Perrine Cruaud
AU  - Jean-Yves Rasplus
AU  - Lillian Jennifer Rodriguez
AU  - Astrid Cruaud
TI  - High throughput sequencing of multiple amplicons for barcoding and integrative taxonomy
AID  - 10.1101/073304
DP  - 2016 Jan 01
TA  - bioRxiv
PG  - 073304
4099  - http://biorxiv.org/content/early/2016/09/04/073304.short
4100  - http://biorxiv.org/content/early/2016/09/04/073304.full
AB  - 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 sequences obtained from the same samplings were similar. For the remaining 5%, species were paraphyletic or the sequences clustered into two divergent groups (>7%) on the final trees (Sanger + MiSeq). These problematic cases are difficult to explain but may represent coding NUMTS or heteroplasms. These results highlight the importance of performing quality control steps, working with expert taxonomists and using more than one marker for DNA-taxonomy or species diversity assessment. The power and simplicity of this method appears promising to build on existing experience, tools and resources while taking advantage of NGS.