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首页> 美国卫生研究院文献>Journal of Biomolecular Techniques : JBT >Universal NGS Library Preparation on the Apollo 324 TM System: Automated Ion Torrent Personal Genome Machine Library Preparation
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Universal NGS Library Preparation on the Apollo 324 TM System: Automated Ion Torrent Personal Genome Machine Library Preparation

机译：在Apollo 324 TM系统上进行通用NGS库准备：自动离子激流个人基因组机库准备

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摘要

The Ion Torrent™ Personal Genome Machine™ (PGM™) is revolutionizing the sequencing world with its “Ion Semiconductor Sequencing”. However, its manual library preparation workflow presents a bottleneck. The Apollo 324™ is a benchtop instrument that fully automates the key steps of the Ion Fragment Library Preparation processing using the PrepXTM PGM Library preparation kit. After a 15-minute setup of reagents on the instrument, the end repair, adapter ligation and size selection are all performed with walk-away operation in just under 3.5 hours. The basic workflow is as follows:Sample Preparation: Raw DNA samples are sheared using the recommended Covaris® settings to provide a 1 μg (15 mL) sheared DNA sample with a broad size distribution centered at around 200 bp.Library Preparation: The sheared DNA, reagents, disposables, and AMPure® XP beads are loaded onto the Apollo 324 instrument work surface. The end repair, adapter ligation and size selection are all performed in less than 3.5 hours using AMPure XP beads for both clean-up and size selection. As many as eight library samples can be prepared per run, and two runs can be performed per day.Library Fragment Quality Assessment: Proper size selection is confirmed by running an Agilent® High Sensitivity Chip. The initial version of the protocol prepares a library fragment with 80% of the total sample between 150-250 bp and an average size of 195 bp, eliminating the need for time-consuming gel size selection. Nick translation and enrichment PCR are typically performed. The resulting DNA library has comparable sequencing results to libraries prepared using the Caliper LabChip® XT system.
机译：离子洪流™个人基因组机™（PGM™）凭借其“离子半导体测序”正在彻底改变测序领域。但是，其手动库准备工作流程存在瓶颈。 Apollo 324™是一款台式仪器，可使用PrepXTM PGM库制备套件完全自动化离子片段库制备处理的关键步骤。在仪器上放置15分钟的试剂后，只需不到3.5个小时的无人值守操作即可进行末端修复，接头连接和大小选择。基本工作流程如下：样品制备：使用推荐的Covaris®设置对原始DNA样品进行剪切，以提供1μg（15 mL）剪切的DNA样品，其大小分布集中在200 bp左右。 ，试剂，一次性用品和AMPure®XP珠粒已加载到Apollo 324仪器的工作面上。使用AMPure XP磁珠进行清理和尺寸选择，都可以在不到3.5小时的时间内完成末端修复，接头连接和尺寸选择。每次运行最多可准备八个文库样品，每天可进行两次运行。库片段质量评估：通过运行Agilent®高灵敏度芯片，可以确定正确的大小选择。该协议的初始版本准备了一个文库片段，其总样本的80％在150-250 bp之间，平均大小为195 bp，从而无需进行费时的凝胶大小选择。通常进行切口平移和富集PCR。所得的DNA文库具有与使用CaliperLabChip®XT系统制备的文库相当的测序结果。

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