Gene library deep sequencing for protein superfamily profiling

Abstract Unravelling protein function at scale remains challenging. Gain-of-function (GOF) screens harnessing large open reading frame (ORF) libraries enable systematic functional annotations of cell behavior drivers and drug targets. Currently, few sequencing methods are established to analyze existing ORF libraries and facilitate future library generation. Here, we developed a barcode-free deep sequencing method termed pooled plasmid library sequencing (PPLseq). PPLseq achieved high efficiency and single nucleotide accuracy, including the detection of previously undocumented variants, on a prototypical super-family ORF library (>300 human G-protein-coupled receptors (GPCRs)). We next combined PPLseq with high-throughput gene engineering to generate a new library of 246 GPCR-fluorescent protein (FP) fusions. We quantified expression of all members of this library and identified robustly expressed yet understudied receptors. Collectively, we demonstrate accessible, scalable, and sensitive ORF library sequencing towards a deeper understanding of proteome function.