Synopsis

The regtools junctions annotate command is a tool to annotate the observed junctions with respect to a known transcript structure. The known transcript structure is in the form of a GTF file obtained from one of the standard Gene Annotation databases such as Ensembl/RefSeq/UCSC etc. The goal of the annotation step is to help identify novel/unusual junctions.

Usage

regtools junctions annotate [options] junctions.bed ref.fa annotations.gtf

Input

Input Description
junctions.bed The BED file with the junctions that have be annotated. This file has to be in the BED12 format. One recommended way of obtaining this file is by running regtools junctions extract. See here for more details.
ref.fa The reference FASTA file. The donor and acceptor sequences used in the "splice-site" column are extracted from the FASTA file.
annotations.gtf The GTF file specifies the transcriptome that is used to annotate the junctions. For examples, the Ensembl GTFs for release78 are here

Options

Option Description
-E Do not skip single exon genes. The default is to skip the single exon genes while annotating junctions.
-o File to write output to. STDOUT by default. The output format is described here
-h Display help message for this command.

Output

Column name Description
chrom Chromosome of the junction.
start Junction start co-ordinate. [zero based format]
end Junction end co-ordinate. [zero based format]
name Identifier for the junction.
score The number of reads supporting the junction. [integer]
strand The strand the junction is identified. Same as the input file. [+/-]
splice_site The two basepairs at the donor and acceptor sites separated by a hyphen. [e.g CT-AG]
acceptors_skipped Number of known acceptors skipped by this junction according to the GTF. SeeNotes below for explanation. [integer]
exons_skipped Number of known exons skipped by this junction according to the GTF. SeeNotes below for explanation. [integer]
donors_skipped Number of known donors skipped by this junction according to the GTF. SeeNotes below for explanation. [integer]
anchor Field that specifies the donor, acceptor configuration. See Notes below for explanation. [D/A/DA/NDA/N]
known_donor Is the junction-donor a known donor in the GTF file? [0/1]
known_acceptor Is junction-donor a known acceptor in the GTF file? [0/1]
known_junction Does the junction have a known donor-acceptor pair according to the GTF file. This is equivalent to "DA" in the "anchor" column.
transcripts The transcripts that overlap the junction according to the input GTF file.
genes The genes that overlap the junction according to the input GTF file.

Notes

Annotating observed junctions with known donor/acceptor/junction information

It is useful to annotate the ends of junction with respect to known acceptors, donors and junctions in the transcriptome. The known acceptor, donor and junction information is computed from the GTF file and this information is then used to annotate the observed junctions.

The junctions are annotated using the following nomenclature(and as shown in the figure below.)

  1. DA - This exon-exon junction is present in the transcriptome provided by the user(GTF) The ends of this junction are hence known donor and known acceptor sites according to the GTF file.

  2. NDA - This exon-exon junction is not present(novel) in the transcriptome provided by the user(GTF) The ends of this junction are known donor and known acceptor sites according to the GTF file.

  3. D - This exon-exon junction is not present(novel) in the transcriptome provided by the user(GTF) The donor of this junction is a known donor but the acceptor is novel.

  4. A - This exon-exon junction is not present(novel) in the transcriptome provided by the user(GTF) The acceptor of this junction is a known acceptor but the donor is novel.

  5. N - This exon-exon junction is not present(novel) in the transcriptome provided by the user(GTF) The ends of this junction are hence not known donor/acceptor sites according to the GTF file.

Anchor-annotation example

Annotating a junction with number of donors/acceptors/exons skipped

Exon skipping is a form of RNA splicing that can be identified using RNAseq data. It is hence useful to compute for every observed putative exon-exon junction, the number of exons skipped, the number of known donor sites skipped and the number of known acceptor sites skipped. The known exons, donors and acceptors are calculated from the user supplied GTF file.

In the example shown below, the observed junction has skipped 2 known donor sites and 2 known acceptor sites. For the number of exons skipped we consider two situations. The second exon in all the three transcripts overlap, if these overlapping exons are merged together, the number of exons skipped is just 1. If these three exons are considered to be different the number of exons skipped is 3. We try and provide both these annotations.

Junction-annotation example

If any of the examples are not clear or if you would like more information please feel free to open an issue on GitHub here or post on the discussion page here.