Long-read sequencing to detect full-length protein-protein interactions
Given the increased predictions on interactome size and demand for protein function information, methods for detecting protein-protein interactions remain a significant development area. The all-vs.-all sequencing (AVA-Seq) method utilizes a convergent fusion plasmid design to make two-hybrid technology amenable to next-generation sequencing. Here, we further innovate to take advantage of synthetic DNA technologies and Oxford Nanopore Technologies long-read sequencing improvements to allow us to determine full-length protein-protein interactions. Here, using this approach we recovered 159 protein-protein interactions from a set of 57 human proteins using multiple forms of validation. Further, when referencing a human gold standard set of interactions, eight full-length protein-protein interactions were recovered from an expected 28 interaction pairs (28.6%), a typical recovery rate for two-hybrid technologies. The AVA-Seq, in combination with the ease of synthetic DNA production and the MinION platform, offers a low-cost, high-throughput alternative for determining protein-protein interactions, which can be utilized in research labs at all stages. 3 Key PointsO_LIFirst application of long-read sequencing for full-length protein-protein interaction studies. C_LIO_LIThe recovery rate of the AVA-Seq method using full-length proteins is on par with other leading methods. C_LIO_LIAdvances in synthetic biology and sequencing technologies make full-length protein interactomes affordable and accessible. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/586447v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1d81412org.highwire.dtl.DTLVardef@10dbb19org.highwire.dtl.DTLVardef@76a04borg.highwire.dtl.DTLVardef@15baf0_HPS_FORMAT_FIGEXP M_FIG C_FIG