Interpreting epistatic interactions is crucial for understanding evolutionary dynamics of complex genetic systems and unveiling structure and function of genetic pathways. Although high resolution mapping of en masse variant libraries renders molecular biologists to address genotype-phenotype relationships, long-read sequencing technology remains indispensable to assess functional relationship between mutations that lie far apart. Here, we introduce JigsawSeq for multiplexed sequence identification of pooled gene variant libraries by combining a codon-based molecular barcoding strategy and de novo assembly of short-read data. We first validate JigsawSeq on small sub-pools and observed high precision and recall at various experimental settings. With extensive simulations, we then apply JigsawSeq to large-scale gene variant libraries to show that our method can be reliably scaled using next-generation sequencing. JigsawSeq may serve as a rapid screening tool for functional genomics and offer the opportunity to explore evolutionary trajectories of protein variants.
Bibliographical noteFunding Information:
This work was supported by the Intelligent Synthetic Biology Center of Global Frontier Project (NRF-2012M3A6A8053632) and by the Pioneer Research Center Program (NRF-2012-0009557) through the National Research Foundation of Korea funded by the Ministry of Science, ICT & Future Planning. This research was also supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI13C2163). We thank Tom Muir’s group for their kind donation of the Intein vector, and member of Bang Lab for their critical comments during this work. We also thank J. Jung for critical comments on the manuscript and E. Kwon for helping gene assembly experiment.
All Science Journal Classification (ASJC) codes
- Biochemistry, Genetics and Molecular Biology(all)
- Physics and Astronomy(all)