Department of Biotechnology and Bioengineering, Sandia National Laboratories, Livermore, California, USA.
PLoS One. 2013 Jul 22;8(7):e68988. doi: 10.1371/journal.pone.0068988. Print 2013.
Next-generation sequencing (NGS) is emerging as a powerful tool for elucidating genetic information for a wide range of applications. Unfortunately, the surging popularity of NGS has not yet been accompanied by an improvement in automated techniques for preparing formatted sequencing libraries. To address this challenge, we have developed a prototype microfluidic system for preparing sequencer-ready DNA libraries for analysis by Illumina sequencing. Our system combines droplet-based digital microfluidic (DMF) sample handling with peripheral modules to create a fully-integrated, sample-in library-out platform. In this report, we use our automated system to prepare NGS libraries from samples of human and bacterial genomic DNA. E. coli libraries prepared on-device from 5 ng of total DNA yielded excellent sequence coverage over the entire bacterial genome, with >99% alignment to the reference genome, even genome coverage, and good quality scores. Furthermore, we produced a de novo assembly on a previously unsequenced multi-drug resistant Klebsiella pneumoniae strain BAA-2146 (KpnNDM). The new method described here is fast, robust, scalable, and automated. Our device for library preparation will assist in the integration of NGS technology into a wide variety of laboratories, including small research laboratories and clinical laboratories.
下一代测序（NGS）技术正在成为一种强大的工具，可用于阐明各种应用的遗传信息。不幸的是，NGS 的迅速普及并没有伴随着自动化技术的改进，以便准备格式化的测序文库。为了解决这一挑战，我们开发了一种用于制备 Illumina 测序分析用测序器就绪 DNA 文库的微流控系统原型。我们的系统将基于液滴的数字微流控（DMF）样品处理与外围模块相结合，创建了一个完全集成的、从样品到文库的平台。在本报告中，我们使用我们的自动化系统从人类和细菌基因组 DNA 样本中制备 NGS 文库。从 5ng 总 DNA 在设备上制备的 E. coli 文库在整个细菌基因组上产生了极好的序列覆盖度，与参考基因组的比对率>99%，甚至基因组覆盖度和良好的质量分数。此外，我们还对以前未测序的多药耐药肺炎克雷伯菌（KpnNDM）BAA-2146 株进行了从头组装。这里描述的新方法快速、稳健、可扩展且自动化。我们的文库制备设备将有助于将 NGS 技术集成到各种实验室中，包括小型研究实验室和临床实验室。
