Rapid and effective isolation of candidate sequences for development of microsatellite markers in 30 fish species by using kit-based target capture and multiplexed parallel sequencing
Rapid and effective isolation of candidate sequences for development of microsatellite markers in 30 fish species by using kit-based target capture and multiplexed parallel sequencing
复制标题
通过使用基于试剂盒的目标捕获和多重并行测序，快速有效地分离候选序列，以开发 30 种鱼类的微卫星标记
DOI：
10.1007/s12686-017-0699-z
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发表时间：
2017
影响因子：
1.1
通讯作者：
Mutsumi Nishida
中科院分区：
文献类型：
--
作者：
Hirohiko Takeshima;Nozomu Muto;Yasuyuki Sakai;Naoya Ishiguro;Keiichiro Iguchi; Satoshi Ishikawa;Mutsumi Nishida
下一代测序（NGS）技术的最新进展加速了野生动物保护遗传学微卫星标记的开发。虽然基于NGS发现含有微卫星的序列，微卫星富集文库测序比全基因组鸟枪法测序更有效，但构建微卫星富集文库的过程有些复杂。因此，许多研究人员更喜欢使用外部服务来进行微卫星富集，这需要更多的时间。为了促进新型微卫星标记的快速有效开发，我们尝试简化构建用于多重并行测序的微卫星富集文库的过程。为了捕获含有微卫星的序列，我们应用了易于使用的市售试剂盒进行杂交和洗涤步骤。在为NGS准备了30种鱼类的鸟枪文库后，我们使用市售试剂盒直接从鸟枪文库中捕获了含有微卫星的DNA片段。接下来，在 454 GS Junior 平台上进行了三轮多重平行测序。每个物种的最终序列都包含高比例的含微卫星序列（从 46% 到 79%）。因此，为每个物种有效地设计了足够数量的引物组，范围从 1029 到 6606。微卫星捕获和测序在一周左右完成，所需时间大大缩短。为了验证我们策略的有效性，我们筛选了 44 个为香鱼 (Plecoglossus altivelis) 设计的潜在引物组。多态性结果表明，23 个标记的等位基因变异将有助于研究群体结构。这些结果证明了我们改进的微卫星标记开发方法的有效性。
Recent advances in next-generation sequencing (NGS) technology have accelerated the development of microsatellite markers for wildlife conservation genetics. Although the discovery of microsatellite-containing sequences based on NGS is more efficient with sequencing of a microsatellite-enriched library than with whole-genome shotgun sequencing, the process of constructing a microsatellite-enriched library is somewhat complicated. Therefore, many researchers prefer to use external services for the microsatellite-enrichment, which requires more time. To facilitate the rapid and effective development of novel microsatellite markers, we attempted to simplify the process of constructing a microsatellite-enriched library for multiplexed parallel sequencing. To capture microsatellite-containing sequences, we applied an easy-to-use commercially available kit for the hybridization and wash steps. After preparing shotgun libraries of 30 fish species for NGS, we captured microsatellite-containing DNA fragments directly from the shotgun libraries by using the commercially available kit. Next, three runs of multiplexed parallel sequencing were conducted on the 454 GS Junior platform. The resulting sequences for each species included high proportions of microsatellite-containing sequences (from 46 to 79%). Thus, sufficient numbers of primer sets, ranging from 1029 to 6606, were effectively designed for each species. Microsatellite capture and sequencing were completed in about a week, so the time required was substantially reduced. To validate the effectiveness of our strategy, we screened 44 potential primer sets designed for ayu (Plecoglossus altivelis). The results of polymorphisms revealed that allelic variability at 23 markers will be useful for studying population structure. These results prove the effectiveness of our improved approach for microsatellite marker development.