Highly efficient library preparation for Ion Torrent sequencing using Y-adapters
Highly efficient library preparation for Ion Torrent sequencing using Y-adapters
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DOI：
10.2144/btn-2019-0035
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发表时间：
2019-11-01
期刊：
影响因子：
2.7
通讯作者：
Hoeper, Dirk
中科院分区：
文献类型：
--
作者：
Forth, Leonie F.;Hoeper, Dirk
文库制备是下一代测序工作流程的关键步骤。文库制备成功的关键决定因素是输入DNA的可用量和将DNA转化为功能文库分子的效率。虽然离子激流库的标准钝端连接协议的理论最高效率为25%，但y适配器通过(i)粘端连接和（ii）使两个DNA链都具有测序功能，从而实现高效的库制备，从而导致理论效率高达100%。此外，减少了适配器二聚体的生成。因此，我们设计、优化和验证了与Ion Torrent测序兼容的y适配器。这些有助于提高文库产量，并结合在关键特征读取长度、碱基质量和文库复杂性方面的总体高测序性能。我们开发、优化和验证了用于Ion Torrent测序的y -适配器，该适配器既非市售也未在其他地方描述过。这些y型适配器通过(i)粘端连接和（ii）呈现两条DNA链，实现了高效的文库制备，因此理论上高达100%的输入DNA链可用于测序。此外，在低DNA输入的情况下，当使用所描述的y -适配器时，适配器二聚体的部分大大减少。
Library preparation is a crucial step in next-generation sequencing workflows. Key determinants of successful library preparation are the available amount of input DNA and the efficiency of the conversion of this DNA into functional library molecules. While the standard blunt-end ligation protocol for Ion Torrent libraries has a theoretical maximum efficiency of 25%, Y-adapters enable highly efficient library preparation by (i) sticky-end ligation and (ii) rendering both DNA strands functional for sequencing, hence resulting in a theoretical efficiency of up to 100%. Moreover, the generation of adapter dimers is reduced. Therefore, we designed, optimized and validated Y-adapters compatible with Ion Torrent sequencing. These facilitate higher library yields combined with overall high sequencing performance regarding the key characteristics read-length, base quality, and library complexity.METHOD SUMMARYWe developed, optimized, and validated Y-adapters for Ion Torrent sequencing, which are neither commercially available nor have been described elsewhere before. These Y-adapters enable highly efficient library preparation by (i) sticky-end ligation and (ii) rendering both DNA strands, hence theoretically up to 100% of the input DNA strands are functional for sequencing. Moreover, with low DNA input, the portion of adapter dimers is substantially reduced when using the described Y-adapters.