Development of ONT-cappable-seq to unravel the transcriptional landscape of Pseudomonas phages

Transcriptomics-driven characterisation of novel T7-like temperate Pseudomonas phage LUZ100

“…To gain more insights in the transcriptional scheme and gene regulation mechanisms of T7-like phages with temperate elements, we studied the viral transcriptome of LUZ100 using ONT-cappable-seq. In contrast to classic RNA sequencing approaches, ONT-cappable-seq allows end-to-end sequencing of primary transcripts and the identification of key regulatory elements in dense phage transcriptomes, such as transcription initiation and termination sites, as well as operon structures (16). To explore the phage transcriptome in a global fashion, RNA from multiple timepoints throughout LUZ100 infection of its P. aeruginosa PaLo41 host were pooled together and sequenced on a MinION sequencing device.…”

“…The overall performance of the sequencing run and raw read quality was assessed using NanoComp (v1.11.2). Next, raw reads were processed and subsequently mapped to the genomes of Pseudomonas phage LUZ100 and P. aeruginosa host strain PaLo41, as described previously (16). Sequencing quality, read lengths and mapping metrics are reported in Supplementary Table S2.…”

“…Alignments were visualized in Integrative Genomics Viewer (IGV) (31). Finally, data analysis was performed according to the ONT-cappable-seq workflow (https://github.com/LoGT-KULeuven/ONT-cappable-seq) to identify viral transcriptional start sites (TSS) and termination sites (TTS) and elucidate the transcriptional architecture of LUZ100 (16). To discriminate between phage-specific and host-specific promoter sequences, regions upstream the annotated TSSs (−100 to +1) were analyzed using MEME and the Pseudomonas σ70 promoter prediction tool SAPPHIRE.CNN (32, 33).…”

“…Genetic features located on the same strand annotated into a TU in case they were covered by at least 90% (bedtools intersect - F 0.9 -s). Based on the TUs and their gene content, complex operon structures were delineated by finding overlapping TUs with the same orientation that share at least one annotated gene (16, 36).…”

“…This phage is, to the best of our knowledge, the first representative of its kind that can infect the pathogen P. aeruginosa . We characterised its morphological and biological properties, performed a genomic analysis and charted the LUZ100 transcriptional landscape using the recently established ONT-cappable-seq method (16). This nanopore-based RNA sequencing method enables full-length profiling of primary prokaryotic transcripts and provides a global map of key viral regulatory sequences involved in transcriptional reprogramming of the host cell.…”