High-throughput cDNA sequencing has emerged as a powerful tool to globally assess the transcriptional state of cells. However post-transcriptional processing and modification events add layers of complexity to transcriptomes that are typically not revealed by standard RNA-seq technologies. Standard RNA-seq confounds the identification of key transcriptional events such as the start and end of transcription and, more generally, the original composition of primary transcripts. Thus, being able to de couple the primary transcriptome from processed RNA is key to determining the association between the regulatory state of the genome and its phenotypic outcome.

Cappable-seq is a novel method that enables direct modification and identification of the triphosphorylated RNA characteristic of primary transcripts. Cappable-seq can discriminates the 5′ triphosphate end characteristic of initiating 5′ triphosphorylated (ppp) nucleotide incorporated by the RNA polymerases from the processed 5′ monophosphate RNAs. The study demonstrates the ability of Cappable-seq to determine TSS at one base resolution genome-wide by pairing Cappable-seq with direct 5′ ligation of sequencing adaptors to the RNA. In addition to and beyond TSS determination, Cappable-seq depletes ribosomal RNA and reduces the complexity of the transcriptome to a single quantifiable tag per transcript enabling digital profiling of gene expression in any microbiome.

Fig.1 Schema of Cappable-seq protocol and the associated control library. (Ettwiller, 2016)

Library Construction of Cappable-Seq

The library construction of Cappable-seq involves several steps, including RNA extraction, mRNA selection, decapping and phosphorylation, linker ligation, cDNA synthesis, PCR amplification, size selection, library preparation, sequencing, and data analysis. Each step is critical for obtaining a high-quality library and accurate TSS identification. The method is highly sensitive and can detect low-abundance transcripts, as well as novel TSSs. Additionally, it enables the study of gene expression regulation and transcriptome dynamics.

Project Workflow

Data analysis

• Gene annotations are derived from the NCBI
• Annotation selecting entries with feature tRNA or rRNA
• Trimmed reads mapping to each position on the genome
• Distribution of the strength of the TSS
• Determination of transcriptional units
• Motifs logo were done using the weblogo3 program
• Go enrichment analysis

Deliverable: FastQ, BAM, QC report, TSS landscape, custom bioinformatics analysis.

References:

1. Ettwiller, Laurence, et al. A novel enrichment strategy reveals unprecedented number of novel transcription start sites at single base resolution in a model prokaryote and the gut microbiome. BMC genomics 17.1 (2016): 1-14.
2. Yan, Bo, et al. SMRT-Cappable-seq reveals complex operon variants in bacteria. Nature communications 9.1 (2018): 1-11.
3. Putzeys, Leena, et al. Development of ONT-cappable-seq to unravel the transcriptional landscape of Pseudomonas phages. Computational and Structural Biotechnology Journal (2022).