ISSRseq: An extensible method for reduced representation sequencing
ISSRseq: An extensible method for reduced representation sequencing
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ISSRseq：一种用于简化表示测序的可扩展方法
DOI：
10.1111/2041-210x.13784
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发表时间：
2021
影响因子：
6.6
通讯作者：
Barrett, Craig F.
中科院分区：
文献类型：
--
作者：
Sinn, Brandon T.;Simon, Sandra J.;Santee, Mathilda V.;DiFazio, Stephen P.;Fama, Nicole M.;Barrett, Craig F.
生成密集采样的单核苷酸多态性（SNP）数据的能力在生物学的不同子学科中是必不可少的，包括作物育种、病理学、法医学、林业、生态学、进化和保护。然而，进行基因组规模变异发现所需的湿实验室专业知识和生物信息学培训仍然是资源有限的研究人员的限制因素。在这里，我们介绍了ISSRseq，一种基于PCR的方法，使用简单重复序列作为引物位点对简单重复序列（ISSR）区域进行测序，以减少基因组变异的代表性。简言之，用单引物扩增ISSR区域，合并，用于用市售试剂盒构建测序文库，并在Illumina平台上测序。我们还提出了一个灵活的生物信息学管道，组装ISSR基因座，调用和硬过滤器变体，以常见格式输出数据矩阵，并使用R进行群体分析。使用三种被子植物物种作为案例研究，我们证明ISSRseq具有高度可重复性，仅需要简单的湿实验室技能和普通仪器，在使用的单引物数量方面灵活，ISSRseq是对任何生物体进行SNP基因分型的一种直接方法，我们预测这种方法对于研究非模式生物的群体基因组学和生物地理学特别有用。此外，ISSRseq相对于其他RRS方法的易用性应该证明对那些缺乏湿实验室方法或生物信息学方面的先进专业知识的人有用。
The capability to generate densely sampled single nucleotide polymorphism (SNP) data is essential in diverse subdisciplines of biology, including crop breeding, pathology, forensics, forestry, ecology, evolution and conservation. However, the wet‐laboratory expertise and bioinformatics training required to conduct genome‐scale variant discovery remain limiting factors for investigators with limited resources.Here we present ISSRseq, a PCR‐based method for reduced representation of genomic variation using simple sequence repeats as priming sites to sequence inter simple sequence repeat (ISSR) regions. Briefly, ISSR regions are amplified with single primers, pooled, used to construct sequencing libraries with a commercially available kit, and sequenced on the Illumina platform. We also present a flexible bioinformatic pipeline that assembles ISSR loci, calls and hard filters variants, outputs data matrices in common formats, and conducts population analyses using R.Using three angiosperm species as case studies, we demonstrate that ISSRseq is highly repeatable, necessitates only simple wet‐laboratory skills and commonplace instrumentation, is flexible in terms of the number of single primers used, and can generate genomic‐scale variant discovery on par with existing RRS methods which require more complex wet‐laboratory procedures.ISSRseq represents a straightforward approach to SNP genotyping in any organism, and we predict that this method will be particularly useful for those studying population genomics and phylogeography of non‐model organisms. Furthermore, the ease of ISSRseq relative to other RRS methods should prove useful to those lacking advanced expertise in wet‐laboratory methods or bioinformatics.