Massively parallel multiplex DNA sequencing for specimen identification using an Illumina MiSeq platform

Genetic information is a valuable component of biosystematics, especially specimen identification through the use of species-specific DNA barcodes. Although many genomics applications have shifted to High-Throughput Sequencing (HTS) or Next-Generation Sequencing (NGS) technologies, sample identification (e.g., via DNA barcoding) is still most often done with Sanger sequencing. Here, we present a scalable double dual-indexing approach using an Illumina Miseq platform to sequence DNA barcode markers. We achieved 97.3% success by using half of an Illumina Miseq flowcell to obtain 658 base pairs of the cytochrome c oxidase I DNA barcode in 1,010 specimens from eleven orders of arthropods. Our approach recovers a greater proportion of DNA barcode sequences from individuals than does conventional Sanger sequencing, while at the same time reducing both per specimen costs and labor time by nearly 80%. In addition, the use of HTS allows the recovery of multiple sequences per specimen, for deeper analysis of genetic variation in target gene regions.