A novel NGS-based method can improve the efficiency and sensitivity of Y2H screening with cDNA library

The NGS-based Y2H screening methods have been adopted to increase the efficiency and sensitivity, while reducing the labor and experimental cost of the canonical Y2H screening. However, most of NGS-based Y2H screening methods are suitable for well-constructed ORFeomes but not cDNA libraries. Thus, we developed a novel NGS-based Y2H screening method to accomplish a precise Y2H screening with cDNA libraries. With newly designed primers, we can distinguish and filter out those non-in-frame reads from all mapped reads, which facilitates the estimation of the interaction intensity between baits and preys. The prey inserts were PCR-amplified from selected yeast colonies (>3000 colonies per sample). After fragmentation, the DNA fragment of prey inserts were ligated to standard Illumina adaptor. Then, the second round PCR amplification was performed to enrich the DNA fragments with 29 bp vector sequence and simultaneously the standard Illumina DNA library for paired-end sequencing was obtained.