Liu Kai, Xie Nan
Institute of Fishery Science, Hangzhou Academy of Agricultural Sciences, Hangzhou, Zhejiang China.
3 Biotech. 2022 Oct;12(10):248. doi: 10.1007/s13205-022-03313-0. Epub 2022 Aug 26.
Microsatellites, also known as simple sequence repeats (SSRs), are the preferred type of marker for many genetic applications. In conjunction with the ongoing development of next-generation sequencing, several bioinformatic tools have been developed for identifying SSRs from genomic or transcriptomic sequences. Although these tools are handy for generating polymorphic SSRs, their application almost always depends on an existing reference genome or self-assembly of the reference genome. With this in mind, we propose a pipeline for developing polymorphic SSRs that may be applied to species without reference genomes. Using a species without a reference genome (black Amur bream; Richardson, 1846) as a model, our pipeline was able to effectively discover polymorphic SSRs. Under different R parameters of a reference-free single nucleotide polymorphisms (SNPs) caller (ebwt2InDel), a total of 258, 208, 102, and 11 polymorphic SSRs were mined. To quantify the accuracy of the polymorphic SSRs detected using our pipeline, we analyzed 25 SSRs with PCR experiments. All primers were successfully amplified, and most SSRs (23 SSRs, 92%) were polymorphic. From the 36 individual black Amur bream, we acquired an average of 3.36 alleles per locus, ranging from one to 11. This demonstrates the effectiveness of our pipeline in identifying polymorphic SSRs and designing primers for SSR genotyping. Ultimately, our pipeline can effectively mine polymorphic SSRs for species without reference genomes, complementing SSR mining approaches based on reference genomes and helping to resolve biological issues that accompany these methods.
The online version contains supplementary material available at 10.1007/s13205-022-03313-0.
微卫星，也称为简单序列重复（SSR），是许多基因应用中首选的标记类型。随着下一代测序技术的不断发展，已经开发了几种生物信息学工具来从基因组或转录组序列中识别SSR。尽管这些工具便于生成多态性SSR，但其应用几乎总是依赖于现有的参考基因组或参考基因组的自我组装。考虑到这一点，我们提出了一种开发多态性SSR的流程，该流程可应用于没有参考基因组的物种。以一种没有参考基因组的物种（黑鲂；Richardson，1846年）为模型，我们的流程能够有效地发现多态性SSR。在无参考单核苷酸多态性（SNP）调用程序（ebwt2InDel）的不同R参数下，共挖掘出258、208、102和11个多态性SSR。为了量化使用我们的流程检测到的多态性SSR的准确性，我们通过PCR实验分析了25个SSR。所有引物均成功扩增，并且大多数SSR（23个SSR，92%）具有多态性。从36条黑鲂个体中，我们在每个位点平均获得3.36个等位基因，范围从1到11个。这证明了我们的流程在识别多态性SSR和设计用于SSR基因分型的引物方面的有效性。最终，我们的流程可以有效地为没有参考基因组的物种挖掘多态性SSR，补充基于参考基因组的SSR挖掘方法，并有助于解决伴随这些方法的生物学问题。
在线版本包含可在10.1007/s13205-022-03313-0获取的补充材料。
