CLIP tags Around Junction Analysis (CAJA)

Investigate patterns of target RNAs bound by RNA Binding Protein (RBP) around splicing junctions from HITS-CLIP data.

CAJA consists of two programs:

• findJunctions.py
• plotFrequency.ipynb

findJunctions.py

Compute the genomic distance between the end/start of HITS-CLIP tags and splicing sites (SS) which are located on both positive and negative genomic DNA strand orientation between a given range (e.g. -50~+50, 0 representing splicing sites).

A parser for command-line options, arguments and sub-commands. Basic usage of findJunctions.v1.7.py :

python3 findJunctions.py splicesites_coordinates.txt HITS-CLIP_RNA_tags.bed -n 50 -o outfile_name

• splicesites_coordinates.txt: tab-delimited text containing splicing sites coordinates which is generated from GTF file by using hisat2_extract_splice_sites.py in hisat2 package. e.g. splicesites_hg38_chr.txt is SS coordinates of human genome hg38.
• HITS-CLIP_RNA_tags.bed: the output bed file of the CTK pipeline for HITS-CLIP data analysis. It provides genomic coordinate information for unique tags bound by RNA-binding proteins.
• -n 50: the given range around splicing sites is -50nt~+50nt.
• -o outfile_name: output files contain four bed-like format files and four summary txt files.

Output files (bed-like files):

The genomic distance between the end/start of tags and splicing sites in a given nucleiotide window. The 1st column: Chromosome; the 2nd column: HITS-CLIP unique tag name; the 3rd column: Coordinates of end/start of tag; the 4th column: Coordinates of 5'SS/3'SS.

• outfile_name.pos.junc.1.bed: HITS-CLIP tags around 5'SS on positive genomic DNA strand orientation, values in the 3rd column are coordinates of the end of tags, and values in the 4th column are coordinates of 5'SS.
• outfile_name.pos.junc.2.bed: HITS-CLIP tags around 3'SS on positive genomic DNA strand orientation, values in the 3rd column are coordinates of the start of tags, and values in the 4th column are coordinates of 3'SS.
• outfile_name.neg.junc.1.bed: HITS-CLIP tags around 3'SS on negative genomic DNA strand orientation, values in the 3rd column are coordinates of the start of tags, and values in the 4th column are coordinates of 3'SS.
• outfile_name.neg.junc.2.bed: HITS-CLIP tags around 5'SS on negative genomic DNA strand orientation, values in the 3rd column are coordinates of the end of tags, and values in the 4th column are coordinates of 5'SS.

Output files (summary txt files):

The summary of the distance between the end/start of tags and splicing sites and their read counts. The following four files are generated based on coordinated in the four bed-like files above, respectively. The 1st column: Count of tags at each site; the 2nd column: Genomic distance between tag and SS, 0 representing SS:

• outfile_name.pos.junc.1.summary.txt
• outfile_name.pos.junc.2.summary.txt
• outfile_name.neg.junc.1.summary.txt
• outfile_name.neg.junc.2.summary.txt

plotFrequency.ipynb

After generating outfiles above, the four summary text files are used for plotting the frequency of occurence of RNA tags bound by the RNA Binding Protein (RBP).

For the plotting around 5'SS, outfile_name.pos.junc.1.summary.txt and outfile_name.neg.junc.2.summary.txt are concatenated:

( cat outfile_name.pos.junc.1.summary.txt ; echo ""; cat outfile_name.neg.junc.2.summary.txt; echo ) > outfile_name.junc.5SS.summary.txt

For the plotting around 3'SS, outfile_name.pos.junc.2.summary.txt and outfile_name.neg.junc.1.summary.txt are concatenated:

( cat outfile_name.pos.junc.2.summary.txt ; echo ""; cat outfile_name.neg.junc.1.summary.txt; echo ) > outfile_name.junc.3SS.summary.txt

The plotting is done using plotFrequency.ipynb in jupyter notebook.

For example, plotFrequency_ALKBH5_FL_C_5SS.ipynb is used for plotting RNA tags bound by ALKBH5 full-length and C-terminus proteins around 5SS; plotFrequency_ALKBH5_FL_C_3SS.ipynb is used for plotting RNA tags bound by ALKBH5 full-length and C-terminus proteins around 3SS. plotFrequency_endo_ALKBH5_AGO2_5SS.ipynb is used for plotting RNA tags bound by endogenous ALKBH5 and AGO2 in A549 cells around 5SS; plotFrequency_endo_ALKBH5_AGO2_3SS.ipynb is used for plotting RNA tags bound by endogenous ALKBH5 and AGO2 in A549 cells around 3SS.