Host-pathogen protein interaction studies: quality control of cDNA libraries using nanopore sequencing

Abstract

Protein-protein interactions (PPI) play a key role in host-pathogens interaction studies, as proteins are essential to many cellular mechanisms. The yeast two-hybrid (Y2H) approach is a well-established method for high-throughput PPI screening and mapping of protein interaction networks. The success of this approach partially depends on the quality and representativeness of the host cDNA library, which can be constructed from the transcriptomic content of a selected host cellular type. However, evaluating the relevance of the cDNA library content remains challenging, and one of the key limitations of this interactomic approach is the occurrence of false-negative results (i.e., the absence of detectable interactions). Here, we report a direct, long read, high-throughput sequencing method using Oxford Nanopore Technologies, to assess the completeness of the host cDNA library used in host-pathogen interactions Y2H screening. This approach enables easy identification of possible downstream screened genes in PPI assays, minimizing sequencing biases and bioinformatics handling of the data. This study was performed on a cDNA library, generated from A549 human lung carcinoma cells. We were able to identify 12,123 protein coding genes from the sequencing of whole plasmids containing the cDNA inserts, that were further analyzed via functional pathways enrichment for deeper characterization. This diversity and relative abundance evaluation method could be a first step when generating new cDNA libraries of interest for PPI studies, ensuring the validity and suitability of the host library before proceeding with all Y2H screening steps.