next - generation sequencing to generate interactome datasets

478 | VOL.8 NO.6 | JUNE 2011 | nature methods and transcriptome ‘shotgun’ sequencing, next-generation DNA sequencing technologies are not readily applicable for identification of interacting pairs. The necessary pooling of PCR amplicons in the preparation of interacting sequence tags (ISTs) (Fig. 1a) would inevitably eliminate the association in each pair of DNA sequences coding for interacting molecules. Here we describe a massively parallel interactome-mapping strategy that incorporates next-generation DNA sequencing (Fig. 1a) and test the strategy in a high-throughput yeast two-hybrid (Y2H) system. This general scheme can be readily extended to increase throughput and decrease cost for other interactome-mapping methods, particularly other binary protein-protein interaction assays1, yeast one-hybrid3 or genetic screens in which pairs of DNA molecules are selected and identified9. In current protocols of high-throughput Y2H screens, the open reading frames (ORFs) or cDNAs encoding selected pairs of interacting hybrid proteins (X fused to a DNA-binding domain (DB-X) and Y fused to an activation domain (AD-Y)) are amplified directly from yeast transformants and subsequently identified by Sanger DNA sequencing4 (Supplementary Fig. 1). As X and Y originate from recorded positions in paired PCR plates, they can be computationally reassembled to form pairs of ISTs10. The first step of our methodology, termed Stitch-seq, is PCR stitching, which places a pair of sequences encoding interacting proteins on the same PCR amplicon11. PCR stitching consists of two rounds of PCR (Fig. 1b). In the first round, X and Y (present on the Y2H DB-X and AD-Y vectors) are amplified with DBand ADvector-specific upstream primers, respectively (Supplementary next-generation sequencing to generate interactome datasets