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Highly Multiplexed, Semiautomated Nextera Next-Generation Sequencing (NGS) Library Preparation
William Christie1, Ron Yadin1, Kristy Ip1
1Amyris Inc., Emeryville, CA, USA.
Methods in Molecular Biology (Clifton, N.J.)
|August 19, 2020
Summary
This study presents a high-throughput DNA sequencing protocol using miniaturized Nextera Tagmentation reactions and custom PCR index primers. This method significantly reduces costs for sequencing thousands of DNA constructs in a single run.
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- High-throughput DNA sequencing is crucial for large-scale DNA assembly.
- Current methods can be costly and time-consuming for massive projects.
Purpose of the Study:
- To develop a cost-effective, high-throughput protocol for DNA sequencing.
- To enable simultaneous sequencing of thousands of DNA constructs.
Main Methods:
- Utilized traditional and acoustic liquid-handling robotics for miniaturization.
- Employed Illumina's Nextera Tagmentation reactions with custom PCR index primers.
- Generated highly multiplexed next-generation sequencing (NGS) libraries for pooled sequencing.
Main Results:
- Successfully miniaturized Nextera Tagmentation reactions.
- Enabled the production of highly multiplexed NGS libraries.
- Achieved simultaneous sequencing of thousands of DNA constructs in a single run.
Conclusions:
- The described protocol offers a dramatically reduced cost for high-throughput DNA sequencing.
- This method is suitable for large-scale DNA assembly operations requiring pooled sequencing.
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