Two sequence-verified, clonal, publicly available collections of human open reading frames are reported. One collection is in a lentiviral vector for expression in mammalian cells; the other is in the Gateway vector system. Functional characterization of the human genome requires tools for systematically modulating gene expression in both loss-of-function and gain-of-function experiments. We describe the production of a sequence-confirmed, clonal collection of over 16,100 human open-reading frames (ORFs) encoded in a versatile Gateway vector system. Using this ORFeome resource, we created a genome-scale expression collection in a lentiviral vector, thereby enabling both targeted experiments and high-throughput screens in diverse cell types.

A combination of PCR stitching with next-generation sequencing results in Stitch-seq, a massively parallel method for interactome mapping. The approach is applied to human protein-protein interactions assayed in yeast two-hybrid screens. Next-generation sequencing has not been applied to protein-protein interactome network mapping so far because the association between the members of each interacting pair would not be maintained in en masse sequencing. We describe a massively parallel interactome-mapping pipeline, Stitch-seq, that combines PCR stitching with next-generation sequencing and used it to generate a new human interactome dataset. Stitch-seq is applicable to various interaction assays and should help expand interactome network mapping.