Library Preparation and Multiplex Capture for Massive Parallel Sequencing Applications Made Efficient and Easy
Library Preparation and Multiplex Capture for Massive Parallel Sequencing Applications Made Efficient and Easy
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DOI：
10.1371/journal.pone.0048616
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发表时间：
2012-11-05
期刊：
影响因子：
3.7
通讯作者：
Klevebring, Daniel
中科院分区：
文献类型：
--
作者：
Neiman, Marten;Sundling, Simon;Klevebring, Daniel
近年来，测序技术的快速发展和竞争激烈的市场使研究人员能够以合理的成本开展大规模测序项目。随着实际测序反应的价格下降，可以对更多样本进行测序，制备文库的相对成本变得更大，实际的实验室工作变得复杂而乏味。我们提出了一种经济高效的策略，用于简化文库制备，与全基因组和靶向测序实验兼容。优化的酶组成和反应缓冲液减少了所需的清理步骤的数量，并允许使用大量酶，这使得整个过程廉价、高效且简单。我们还提出了一种双标记策略，该策略允许对目标区域进行多重测序。为了证明我们的想法，我们准备了用于对 100 ng DNA 进行低通测序的文库，对 500 kb 区域进行了 2、4 和 8 重外显子组捕获以及 96 重捕获。在所有样本中，与市售 SNP 芯片平台相比，我们发现 SNP 识别具有很高的一致性 (>99.4%)。
During the recent years, rapid development of sequencing technologies and a competitive market has enabled researchers to perform massive sequencing projects at a reasonable cost. As the price for the actual sequencing reactions drops, enabling more samples to be sequenced, the relative price for preparing libraries gets larger and the practical laboratory work becomes complex and tedious. We present a cost-effective strategy for simplified library preparation compatible with both whole genome-and targeted sequencing experiments. An optimized enzyme composition and reaction buffer reduces the number of required clean-up steps and allows for usage of bulk enzymes which makes the whole process cheap, efficient and simple. We also present a two-tagging strategy, which allows for multiplex sequencing of targeted regions. To prove our concept, we have prepared libraries for low-pass sequencing from 100 ng DNA, performed 2-, 4- and 8-plex exome capture and a 96-plex capture of a 500 kb region. In all samples we see a high concordance (>99.4%) of SNP calls when comparing to commercially available SNP-chip platforms.