Large-Scale Low-Cost NGS Library Preparation Using a Robust Tn5 Purification and Tagmentation Protocol

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• High-quality sequencing library preparation is crucial for next-generation sequencing (NGS).
• Tn5 transposase offers efficient DNA fragmentation and adapter insertion for library construction.
• Cost-effectiveness and versatility are key considerations for large-scale genomic studies.

Purpose of the Study:

• To present a robust and cost-effective Tn5 transposase purification strategy.
• To develop improved Tn5 enzyme variants for enhanced experimental flexibility.
• To demonstrate the utility of in-house Tn5 enzymes in various sequencing applications.

Main Methods:

• Purification of Tn5 transposase using an N-terminal His6-Sumo3 tag.
• Preparation of sequencing libraries using in-house purified Tn5 enzymes.
• Characterization of Tn5 enzyme variants with specific point mutations (E54K, L372P, R27S).

Main Results:

• In-house Tn5 enzyme produced libraries of comparable quality to commercial kits (Nextera XT).
• The developed strategy significantly reduces costs for large-scale sequencing experiments.
• Two Tn5 variants with distinct fragment size control capabilities were generated and validated.

Conclusions:

• The presented Tn5 purification strategy provides a high-quality, cost-effective alternative for NGS library preparation.
• Improved Tn5 enzyme variants offer enhanced control over fragment size distribution for customized experimental designs.
• The versatile Tn5 enzymes are suitable for diverse applications, including ultralow input and single-cell sequencing.