PolyASite in action
We used the PolyASite resource in our own research and developed three tools that exploit the comprehensive annotation of 3' ends.
PAQR is a method for quantifying the polyadenylation site use (and relative abundance of 3' UTR isoforms) from RNA sequencing data. PAQR identifies annotated poly(A) sites where significant drops in RNA-seq read coverage occur, indicating that 3' end processing occurs at those positions.
KAPAC stands for k-mer activity on polyadenylation site choice and is a method that infers position-dependent activities of sequence motifs on 3'end processing from changes in poly(A) site usage between conditions.
TECtool (Terminal Exon Characterization tool) is a computational method that uses RNA-seq data and the PolyASite atlas to identify novel terminal exons that are located in regions currently annotated as 'intronic'. TECtool infers transcripts that could end at the novel exons and annotates their putative coding regions.
KAPAC can be applied directly to 3' end quantifications that were obtained from data sets outlined in the experiments tab of the PolyASite. To also allow the application of KAPAC on RNA-seq data, PAQR was developed to quantify the use of annotated poly(A) sites (e.g. from PolyASite) from RNA-seq, such that its output can be seamlessly fed into KAPAC. Both tools are available on https://github.com/zavolanlab/PAQR_KAPAC.
TECtool can be easily installed via conda or ran out of the box via docker (or singularity). TECtool takes as input a fasta file containing all chromosomes, a corresponding annotation file in GTF format (e.g. ENSEMBL), a file with genome coordinates of 3’ end processing sites in BED format and a file containing spliced alignments of mRNA-seq reads to the corresponding genome. Find out more on https://github.com/zavolanlab/TECtool or tectool.unibas.ch.
A full use case application of PAQR and KAPAC through a snakemake pipeline is available on zenodo. The available archive contains all necessary input data, scripts and software packages to be started out of the box on a linux systems. It can be downloaded from https://doi.org/10.5281/zenodo.1147433