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 €) was estimated to decrease (< 5.00 €) in studies aiming for a higher throughput. We provide guidelines for selecting NGS or Sanger sequencing depending on the goals of future studies.
KeywordsNGS phylogeny heteroplasmy Halictus smaragdulus Wolbachia
Sequencing and library preparation was performed at the Genomics Core of KU Leuven (Belgium) with the help of Sigrun Jackmaert. We would like to thank the valuable suggestions of the two anonymous reviewers.
GS, ZTN and TB conceived the research and wrote the article. GS and SW collected the data and performed the analyses. AP, ZTN, MV, KJ, JVH and MDM contributed to the interpretation of the data. All authors revised the text and the figures and approved the final manuscript.
This study was funded by the Belgian Science Policy (BELSPO) and supported by the FWO Research Community W0.009.11N’Belgian Network for DNA Barcoding’ (BeBoL).
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