A typical molecular cloning procedure requires Sanger sequencing for sequence validation, which is cost-prohibitive and labor-intensive for large-scale clone analysis in genotype-phenotype studies. Here we present the cost-effective clone analysis platform TnClone, which uses next-generation sequencing based on Tn5 tagmentation to rapidly analyze a large number of clones from cell lysates. This method bypasses the extensive plasmid purification step. We also developed a user-friendly graphical user interface and provided general guidelines for conducting validation experiments. We tested our program with 1023 plasmids (222 from cell lysates and 801 from purified clones) and achieved 92% and 99.3% sensitivity with cell lysates and purified DNA, respectively. Our platform provides rapid turnaround with minimal hands-on time for secondary evaluation, as next-generation sequencing technology continues to evolve.
Bibliographical noteFunding Information:
We would like to thank members of the Bang laboratory for their helpful discussions about the manuscript and for testing the algorithm. We would also like to thank members of the Junho Chung laboratory for kindly donating cloned plasmid vectors carrying the single-chain fragment variable antibody. This work was supported by Midcareer Researcher Program (2018R1A2A1A05079172), Bio & Medical Technology Development Programs (NRF-2016M3A9B6948494 and NRF-2018M3A9H3024850) through the National Research Foundation of Korea funded by the Ministry of Science, ICT & Future Planning and Korea Health Technology R&D Project through the Korea Health Industry Development Institute(KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI18C2282).
© 2019 American Chemical Society.
All Science Journal Classification (ASJC) codes
- Biomedical Engineering
- Biochemistry, Genetics and Molecular Biology (miscellaneous)