For molecular insect identification, amplicon sequencing methods are recommended because they offer a cost-effective approach for targeting small sets of informative genes from multiple samples. In this context, high-throughput multilocus amplicon sequencing has been achieved using the MiSeq Illumina sequencing platform. However, this approach generates short gene fragments of <500 bp, which then have to be overlapped using bioinformatics to achieve longer sequence lengths. This increases the risk of generating chimeric sequences or leads to the formation of incomplete loci. Here, we propose a modified nested amplicon sequencing method for targeting multiple loci from pinned insect specimens using the MiSeq Illumina platform. The modification exists in using a three-step nested PCR approach targeting near full-length loci in the initial PCR and subsequently amplifying short fragments of between 300 and 350 bp for high-throughput sequencing using Illumina chemistry. Using this method, we generated 407 sequences of three loci from 86% of all the specimens sequenced. Out of 103 pinned bee specimens of replicated species, 71% passed the 95% sequence similarity threshold between species replicates. This method worked best for pinned specimens aged between 0 and 5 years, with a limit of 10 years for pinned and 14 years for ethanol-preserved specimens. Hence, our method overcomes some of the challenges of amplicon sequencing using short read next generation sequencing and improves the possibility of creating high-quality multilocus barcodes from insect collections.

中文翻译：

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一种使用MiSeq生成固定昆虫标本的多位点条形码的方法。

对于分子昆虫鉴定，推荐使用扩增子测序方法，因为它们为靶向来自多个样品的少量信息基因提供了一种经济高效的方法。在这种情况下，使用MiSeq Illumina测序平台已实现了高通量多基因座扩增子测序。但是，这种方法会产生<500 bp的短基因片段，然后必须使用生物信息学进行重叠才能获得更长的序列长度。这增加了产生嵌合序列的风险或导致不完整基因座的形成。在这里，我们提出了一种改良的嵌套扩增子测序方法，该方法可使用MiSeq Illumina平台靶向固定昆虫标本中的多个基因座。该修饰存在于在初始PCR中使用三步嵌套PCR方法靶向接近全长基因座的位置，然后使用Illumina化学方法扩增300至350 bp之间的短片段以进行高通量测序。使用此方法，我们从86％的所有标本样本中生成了407个三个基因座的序列。在103个复制物种的固定蜜蜂标本中，有71％通过了物种复制之间95％的序列相似性阈值。此方法最适用于0到5岁之间的固定标本，固定标本的最长期限为10年，乙醇保存标本的最高期限为14年。因此，我们的方法克服了使用短读下一代测序技术进行扩增子测序的挑战，并提高了从昆虫收集物中创建高质量多位点条形码的可能性。