OpenTn5: Open-Source Resource for Robust and Scalable Tn5 Transposase Purification and Characterization.

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Tagmentation is a key method for DNA fragmentation and adapter addition in sequencing library preparation.
• The bacterial Tn5 transposase is central to tagmentation, but its efficient production and application require optimization.

Purpose of the Study:

• To develop and validate an open-source protocol for generating a multi-purpose, hyperactive Tn5 transposase (OpenTn5).
• To benchmark the performance of the OpenTn5 enzyme in various genomics applications.

Main Methods:

• Engineering and production of a hyperactive Tn5 transposase variant (pG-Tn5^{E54K, L372P}) in *E. coli*.
• Benchmarking the enzyme's performance in CUT&Tag, bulk ATAC-seq, and single-cell ATAC-seq assays.
• Assessing the long-term stability and activity of the purified enzyme.

Main Results:

• The OpenTn5 protocol successfully produced multi-milligram quantities of active pG-Tn5^{E54K, L372P} protein per liter of *E. coli* culture.
• The generated enzyme demonstrated high efficiency in tagmentation reactions, sufficient for thousands of applications.
• The enzyme retained its activity for over a year during storage, indicating excellent stability.

Conclusions:

• The OpenTn5 protocol provides a scalable and cost-effective method for producing high-quality, hyperactive Tn5 transposase.
• This open-source resource facilitates widespread adoption of efficient tagmentation-based sequencing library preparation methods.
• The robust and stable nature of OpenTn5 enhances its utility in diverse genomic research, including CUT&Tag and ATAC-seq.