RNA-SeQC result interpretation

Question

[gskbioinfo143]

This is regarding RNA-SeQC result clarification.
when gencode.v7.annotation.gtf was used with command
java -jar RNA-SeQC_v1.1.8.jar -n 84408 -s "DGL1094|DGL-finalsort-104.bam|DGL1094" -t gencode.v7.annotation.gtf -r hg19_.fasta -o ./DGLfinalsort/ -strat gc -gc gencode.v7.gc.txt -BWArRNA human_all_rRNA.fasta -noDoC -singleEnd -transcriptDetails -rRNAdSampleTarget 1 million

thre was no rRNA count in my bam file but when used command

java -jar RNA-SeQC_v1.1.8.jar -n 84408 -s "DGL1094|DGL-finalsort-104.bam|DGL1094" -t gencode.v7.annotation.gtf -r hg19_.fasta -o ./DGLfinalsort/ -strat gc -gc gencode.v7.gc.txt -noDoC -singleEnd -transcriptDetails -rRNAdSampleTarget 1 million

rRNA count was 39462. i want to clarify is the problem with human_all_rRNA.fasta file(downloaded from webpage).

if the gencode.v19.annotation.gtf of hg 19 version was used the rRNA count was different 4506. i just want to clarify wht went wrong?
which gtf file to consider for calculation.

Hope to hear frm you

Thank you

Answer 1

[GouthamAtla]

According to the manual using '-BWArRNA' is more robust method.

If you would like to know about rRNA reads, you could:

1. Pull out the rRNA coordinates from the GTF file in a bed format and see how many reads mapped to those regions ? See their mapping quality stats etc.

2. Align the reads to human_all_rRNA.fasta and see how many reads mapped there ? This should not take long time.

This is what the tool is doing but if you do independently it will give more insights to the problem.