Using next-generation sequencing to improve DNA barcoding: lessons from a small-scale study of wild bee species (Hymenoptera, Halictidae)

The parallel sequencing of targeted amplicons is a scalable application of next-generation sequencing (NGS) that can advantageously replace Sanger sequencing in certain DNA barcoding studies. It can be used to sequence different PCR products simultaneously, including co-amplified products. Here, we explore this approach by simultaneously sequencing five markers (including the DNA barcode and a diagnostic marker of Wolbachia) in 12 species of Halictidae that were previously DNA barcoded using Sanger sequencing. Consensus sequences were obtained from fresh bees with success rates of 74-100% depending on the DNA fragment. They improved the phylogeny of the group, detected Wolbachia infections (in 8/21 specimens) and characterised haplotype variants. Sequencing cost per marker and per specimen (11.43 a,notsign) was estimated to decrease (< 5.00 a,notsign) in studies aiming for a higher throughput. We provide guidelines for selecting NGS or Sanger sequencing depending on the goals of future studies.