Next-generation sequencing to generate interactome datasets

Abstract

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.

Figure 1

Stitch-Seq interactome mapping. (a) Interactome mapping using different sequencing technologies. Above, each DNA fragment within each interacting pair is PCR-amplified individually and Sanger sequenced. The association is tracked via position on the plate. Below, each pair of DNA fragments is placed on the same PCR amplicon by PCR-stitching. The amplicons are then collected and subjected to next-generation sequencing. (b) Outline of a PCR stitching experiment.

Figure 1

Stitch-Seq interactome mapping. (a) Interactome mapping using different sequencing technologies. Above, each DNA fragment within each interacting pair is PCR-amplified individually and Sanger sequenced. The association is tracked via position on the plate. Below, each pair of DNA fragments is placed on the same PCR amplicon by PCR-stitching. The amplicons are then collected and subjected to next-generation sequencing. (b) Outline of a PCR stitching experiment.

Figure 2

Human interactome (“HI-NGS”) produced by massively parallel interactome mapping. (a) ORF Search spaces within the human ORFeome 3.1 space of HI1 (8K × 8K and HI-NGS (6K × 6K). (b) Length distribution of 454 reads for HI-NGS. (c) Overlaps between interactions identified by 454 FLX and Sanger sequencing. (d, e) Fraction of protein pairs in the indicated datasets that test positive by PCA (d) and wNAPPA (e). (f) Length distribution of the ORFs in HI1 and HI-NGS with 454 sequencing or Sanger sequencing.

Figure 3

HI-NGS network. (a) Network view (main connected component above the unconnected components) of HI-NGS (gold) produced with PCR stitching compared to HI1 (blue). (b) Degree distribution of HI-NGS compared to HI1.