Increased Throughput by Parallelization of Library Preparation for Massive Sequencing
Increased Throughput by Parallelization of Library Preparation for Massive Sequencing
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DOI：
10.1371/journal.pone.0010029
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发表时间：
2010-04-06
期刊：
影响因子：
3.7
通讯作者：
Lundeberg, Joakim
中科院分区：
文献类型：
--
作者：
Lundin, Sverker;Stranneheim, Henrik;Lundeberg, Joakim
背景：大规模并行测序系统在数据输出方面不断改进，同时使劳动密集型文库准备成为潜在的瓶颈。目前正在努力减轻为高通量测序准备DNA的关键和耗时的工作。方法/主要发现：在这项研究中，我们展示了一种使用通用羧酸包裹的超顺磁珠和聚乙二醇沉淀的自动化并行文库制备方法，作为一种可重复和灵活的DNA片段长度分离方法。利用这种方法，DNA测序的文库准备可以很容易地调整到所需的片段长度。本文使用GS FLX钛仪器演示的自动化方法与标准的手动文库制备方法进行了比较，显示出更高的产率、吞吐量和极好的重复性。此外，我们还平行准备了12个文库，并对其进行了唯一标记，利用定量聚合酶链式反应辅助汇集，可以改善这些标引样本之间的序列读数分布。结论/意义：我们提出了一种新的自动化程序，可以使每人和每天准备36个索引文库，最多可以增加到96个同时处理的文库。该协议的产量、速度和稳健的性能构成了对目前人工方法的实质性改进，而不需要大量的设备投资。所描述的程序能够显著提高中小型测序中心的效率。
Background: Massively parallel sequencing systems continue to improve on data output, while leaving labor-intensive library preparations a potential bottleneck. Efforts are currently under way to relieve the crucial and time-consuming work to prepare DNA for high-throughput sequencing.Methodology/Principal Findings: In this study, we demonstrate an automated parallel library preparation protocol using generic carboxylic acid-coated superparamagnetic beads and polyethylene glycol precipitation as a reproducible and flexible method for DNA fragment length separation. With this approach the library preparation for DNA sequencing can easily be adjusted to a desired fragment length. The automated protocol, here demonstrated using the GS FLX Titanium instrument, was compared to the standard manual library preparation, showing higher yield, throughput and great reproducibility. In addition, 12 libraries were prepared and uniquely tagged in parallel, and the distribution of sequence reads between these indexed samples could be improved using quantitative PCR-assisted pooling.Conclusions/Significance: We present a novel automated procedure that makes it possible to prepare 36 indexed libraries per person and day, which can be increased to up to 96 libraries processed simultaneously. The yield, speed and robust performance of the protocol constitute a substantial improvement to present manual methods, without the need of extensive equipment investments. The described procedure enables a considerable efficiency increase for small to midsize sequencing centers.