Freshwater vertebrate metabarcoding on Illumina platforms using double-indexed primers of the mitochondrial 16S rRNA gene

• Main Author: Vences, Miguel
• Publication Date: 2016
• Other Authors: Lyra, Mariana L. [UNESP], Perl, R. G. Bina, Bletz, Molly C., Stankovic, David, Lopes, Carla Martins [UNESP], Jarek, Michael, Bhuju, Sabin, Geffers, Robert, Haddad, Celio F. B. [UNESP], Steinfartz, Sebastian
• Format: Article
• Language: eng
• Source: Repositório Institucional da UNESP
• Download full: http://dx.doi.org/10.1007/s12686-016-0550-y; http://hdl.handle.net/11449/161850
• topic: eDNA; Double-indexed primers; Amphibians; Discoglossus; NUMT

Metabarcoding is a promising tool for biodiversity inventories and other applications in conservation genetics. We developed a new pair of primers for efficient and affordable high-throughput analysis of a 250 base pair stretch of DNA from the mitochondrial 16S rRNA gene of vertebrates, especially amphibians and fishes. By adapting a double-indexed protocol for Illumina platforms, our approach allows pooling of hundreds of samples in a single sequencing run. We obtained high detection rates of 82-93 % for fish in two German streams, 70 % for mock mixes of DNA from amphibians and fishes, and could distinguish multiple gene copies in amphibians, probably caused by nuclear-mitochondrial transposed DNA or heteroplasmy.