AbstractThe adoption of Next-Generation Sequencing (NGS) by the field of DNA barcoding of Metazoa has been hindered by the fit between the classical COI barcode and the Sanger-based sequencing method. Here we describe a framework for the sequencing and multiplexing of mitogenomes on NGS platforms that implements (I) a universal long-range PCR-based amplification technique, (II) a two-level multiplexing approach (i.e. divergence-based and specific tag indexing), and (III) a dedicated demultiplexing and assembling script from an Ion Torrent sequencing platform. We provide a case study of mitogenomes obtained for two vouchered individuals of daces Leuciscus burdigalensis and L. oxyrrhis and show that this workflow enables to recover over 100 mitogenomes per sequencing chip on a PGM sequencer, bringing the individual cost down below 7,50€ per mitogenome (as of current 2015 sequencing costs). The use of several kilobases for identification purposes, as involved in the improved DNA-barcode we propose, stress the need for data reliability, especially through metadata. Based on both scientific and economic considerations, this framework presents a relevant approach for multiplexing samples, adaptable on any desktop NGS platform. It enables to extend from the prevalent barcoding approach by shifting from the single COI to complete mitogenome sequencing