School of Agriculture, Food and Wine, The University of Adelaide, Adelaide, SA, Australia.
The Bioinformatics Hub, The University of Adelaide, Adelaide, SA, Australia.
Mol Ecol Resour. 2020 May;20(3). doi: 10.1111/1755-0998.13143. Epub 2020 Mar 6.
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.
对于分子昆虫鉴定，推荐使用扩增子测序方法，因为它们为从多个样本中靶向小的信息基因提供了一种具有成本效益的方法。在这种情况下，使用 MiSeq Illumina 测序平台已经实现了高通量多位点扩增子测序。然而，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将这些片段重叠，以获得更长的序列长度。这增加了产生嵌合序列的风险，或者导致不完全基因座的形成。在这里，我们提出了一种改良的嵌套扩增子测序方法，用于使用 MiSeq Illumina 平台从钉住的昆虫标本中靶向多个基因座。改良之处在于使用三步嵌套 PCR 方法，在初始 PCR 中靶向近全长基因座，然后使用 Illumina 化学法扩增 300-350 bp 之间的短片段进行高通量测序。使用这种方法，我们从 86%测序的标本中生成了三个基因座的 407 个序列。在 103 个重复物种的钉住的蜜蜂标本中，71%的物种重复之间的序列相似性阈值超过 95%。这种方法最适用于年龄在 0-5 岁之间的钉住标本，对于钉住的标本，最长为 10 年，对于乙醇保存的标本，最长为 14 年。因此，我们的方法克服了使用短读长下一代测序进行扩增子测序的一些挑战，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。
