Most RNA footprinting approaches that require ribonuclease cleavage generate RNA fragments bearing a phosphate or cyclic phosphate group at their 3′ end. Unfortunately, current library preparation protocols rely only on a 3′ hydroxyl group for adaptor ligation or poly-A tailing. Here, IMMAGINA BioTechnology researchers developed circAID-p-seq, a PCR-free library preparation for selective 3′ phospho-RNA sequencing. As a proof of concept, they applied circAID-p-seq to ribosome profiling, which is based on sequencing of RNA fragments protected by ribosomes after endonuclease digestion. CircAID-p-seq, combined with the dedicated computational pipeline circAidMe, facilitates accurate, fast and highly efficient sequencing of phospho-RNA fragments from eukaryotic cells and tissues. The researchers used circAID-p-seq to portray ribosome occupancy in transcripts, providing a versatile and PCR-free strategy to possibly unravel any endogenous 3′-phospho RNA molecules.
(A) CircAID-p-seq library preparation: step 1: 5′ phosphorylation of the RNA fragment; step 2: selective capture of the 3′P end through the ligation with the RNA-based adaptor; step 3: circularization; step 4: Reverse Transcription – Rolling Circle Amplification (RT-RCA) – generation of the first strand, and second strand cDNA synthesis; step 5: direct cDNA nanopore sequencing. (B) Bioinformatics pipeline. Bases are called from the ONT output (FAST5 files) and the resulting reads (FASTQ file) are processed with circAidMe, which identifies copies of the RNA fragments and calculates their consensus sequence. Data are filtered of contaminants (e.g. rRNAs, tRNAs, other non-protein-coding transcripts) as needed and aligned against the reference sequences (e.g. genome or transcriptome).
Availability – Source code of CircAidMe along with a pre-compiled package and a description of the software are available on GitHub: https://github.com/ms-gx/CircAidMe.