Data from: Next-generation DNA barcoding: using next-generation sequencing to enhance and accelerate DNA barcode capture from single specimens

DNA barcoding is an efficient method to identify specimens and to detect
undescribed/cryptic species. Sanger sequencing of individual specimens is
the standard approach in generating large-scale DNA barcode libraries and
identifying unknowns. However, the Sanger sequencing technology is, in
some respects, inferior to next-generation sequencers, which are capable
of producing millions of sequence reads simultaneously. Additionally,
direct Sanger sequencing of DNA barcode amplicons, as practiced in most
DNA barcoding procedures, is hampered by the need for relatively
high-target amplicon yield, coamplification of nuclear mitochondrial
pseudogenes, confusion with sequences from intracellular endosymbiotic
bacteria (e.g. Wolbachia) and instances of intraindividual variability
(i.e. heteroplasmy). Any of these situations can lead to failed Sanger
sequencing attempts or ambiguity of the generated DNA barcodes. Here, we
demonstrate the potential application of next-generation sequencing
platforms for parallel acquisition of DNA barcode sequences from hundreds
of specimens simultaneously. To facilitate retrieval of sequences obtained
from individual specimens, we tag individual specimens during PCR
amplification using unique 10-mer oligonucleotides attached to DNA
barcoding PCR primers. We employ 454 pyrosequencing to recover full-length
DNA barcodes of 190 specimens using 12.5% capacity of a 454 sequencing run
(i.e. two lanes of a 16 lane run). We obtained an average of 143 sequence
reads for each individual specimen. The sequences produced are full-length
DNA barcodes for all but one of the included specimens. In a subset of
samples, we also detected Wolbachia, nontarget species, and heteroplasmic
sequences. Next-generation sequencing is of great value because of its
protocol simplicity, greatly reduced cost per barcode read, faster
throughout and added information content.

DNA条形码（DNA barcoding）是一种用于鉴定标本及检测未描述/隐存物种的高效方法。
对单个标本进行桑格测序（Sanger sequencing）是构建大规模DNA条形码文库、鉴定未知标本的标准方案。然而，桑格测序技术在诸多方面逊于可同时产出数百万条序列读段的下一代测序仪（next-generation sequencers）。
此外，多数DNA条形码流程中常规采用的直接对DNA条形码扩增子进行桑格测序的方法，受限于较高的靶标扩增子产量要求、核线粒体假基因的共扩增问题、与细胞内共生细菌（如沃尔巴克氏体属Wolbachia）序列的混淆，以及个体内序列变异（即异质性heteroplasmy）情况。任何此类状况均可能导致桑格测序失败，或生成的DNA条形码存在歧义。
本研究证实了下一代测序平台可用于同时从数百份标本中并行获取DNA条形码序列的潜在应用价值。
为便于从单个标本中回收所得序列，我们在PCR扩增阶段为每份标本添加标记：使用连接于DNA条形码PCR引物的独特10聚体寡核苷酸。
我们借助454焦磷酸测序（454 pyrosequencing），在一次454测序运行12.5%的测序通量下（即16泳道运行中的2个泳道），成功获取了190份标本的全长DNA条形码。
每份标本平均获得143条序列读段。
除1份标本外，其余所有标本均获得了全长DNA条形码序列。
在部分样本中，我们还检测到了沃尔巴克氏体、非靶标物种以及异质性序列。
下一代测序因操作流程简便、单条形码读段成本大幅降低、通量更高且可提供更多信息内容，具备极高的应用价值。