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Percentage of RNA-seq reads in exonic, intronic, intergenic and splice junction regions

Dear all, how could I know proportion of different genomic features for my RNA sequence data? like I want to know that what %age of my data falls in exonic, intronic, intergenic and splice junction regions? I have multiple analysis files that looks like this:

1) genome annotation file that give information about mRNA and CDS:

chr4    GLEAN   mRNA    123284514   123288477   0.999991    -   .   ID=Cotton_A_18927_BGI-A2_v1.0;Name=Cotton_A_18927;source_id=CottonA_GLEAN_10022228;identical_support_id=CUFF67.1103.1;evid_id=Cot030308.1
chr4    GLEAN   CDS 123288376   123288477   .   -   0   Parent=Cotton_A_18927_BGI-A2_v1.0
chr4    GLEAN   CDS 123287662   123287826   .   -   0   Parent=Cotton_A_18927_BGI-A2_v1.0

2) a splice junction determination file that gives information for the doner and accepter splice site coordinates:

chr1    329728  329839  -
chr1    330066  330757  -
chr1    581256  581357

3) a transcriptome assembly that give information about exons and transcript:

chr1    StringTie   transcript  328635  330943  1000    -   .   "gene_id ""STRG.1"""    " transcript_id ""STRG.1.1"""   " cov ""70.023491"""    " FPKM ""28.098141"""   " TPM ""24.855738"""    
chr1    StringTie   exon    328635  329729  1000    -   .   "gene_id ""STRG.1"""    " transcript_id ""STRG.1.1"""   " exon_number ""1"""    " cov ""88.673470"""        
chr1    StringTie   exon    329840  330067  1000    -   .   "gene_id ""STRG.1"""    " transcript_id ""STRG.1.1"""   " exon_number ""2"""    " cov ""22.850203"""        
chr1    StringTie   exon    330758  330943  1000    -   .   "gene_id ""STRG.1"""    " transcript_id ""STRG.1.1"""   " exon_number ""3"""    " cov ""18.054590"""

needs kind help.

thank you so much

rna-seq
• 3,604 views • link
updated by WouterDeCoster WouterDeCoster • written by blooming.daisy333 • blooming.daisy333

2 answers

You can use read_distribution.py from RSeQC for this. It works with a bed format as input.

• 1 views • link
written by Martombo Martombo

(Moved to an answer because seems to solve the question of OP)

• 1 views • link
written by WouterDeCoster WouterDeCoster

thank you so much for the response. I have read read_distribution.py from RSeQC. if I provide it with alignment.sam file and gene_annotation.bed it will give me the read distribution only for the TSS TES exon etc but will not provide my any information that what %age of data consists of splice junctions.

let me repharase what I need: 1) %gae of SJ, % of intron, % of inergenic, % exon

2) what % of splice junction resides in: 5'UTR, 3'UTR-CDS, CDS, CDS-3'UTR, 3'UTR,

kindly guid me in this regard

thank you so much

• 1 views • link
written by blooming.daisy333 • blooming.daisy333

RSeQC has also tools for analysing junctions. It counts and classifies junction into known, partial-known, and novel junctions. You can use this as a starting point for your junction-analysis.

• 1 views • link
written by michael.ante michael.ante

(Moved to an answer because seems to solve the question of OP)

• 1 views • link
written by WouterDeCoster WouterDeCoster

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