exon count

[R adele]

Hello I did  pair-ended rna seq, Im now at the analysis part. I tried to using featurecounts to  get the read count on the exon level. 

The summary of the read counts says there were zero  chimeric reads, which I know is  incorrect , because there  are chimeric reads in our data . Im just wondering why it says zero chimeric reads? here is the following  script I ran.

#!/bin/bash

#PBS -N family_13_01_featureCount

#PBS -l nodes=2:ppn=8

#PBS -l vmem=32g

#PBS -l walltime=48:00:00 

#PBS -joe /hpf/projects/Leon/sequencing_repository/raw_fastq/RNA_tom/bam

#PBS -l mem=32g

set -euxo pipefail

cd /hpf/projects/Leon/sequencing_repository/raw_fastq/RNA_tom/bam

/hpf/tools/centos6/subread/1.5.3/bin/featureCounts -a /hpf/projects/Leon/sequencing_repository/raw_fastq/RNA_tom/bam/gencode.v19.annotation.gtf -o family_13_01_exons_RNA-seq_sorted.counts -p -t exon -s 2 -T 12 -f -O -M D4775_R312_BAligned.sortedByCoord.out.bam D4754_R311_BAligned.sortedByCoord.out.bam D4776_R310Aligned.sortedByCoord.out.bam D4777_R307Aligned.sortedByCoord.out.bam D4778_R308BAligned.sortedByCoord.out.bam D4828_R309_BAligned.sortedByCoord.out.bam 

[Wei Shi]

As you did not specify '-C' option in your command, chimeric fragments in your data were counted. That is the reason why the number of chimeric fragments is zero in the counting summary. The counting summary gives you numbers of fragments that were NOT counted due to different filters, in addition to the total number of counted fragments.

[R adele]

Wei Shi  I  wanted to see what would happen to my read counts if I used -C option. It still says in the summary report  that Unassigned_Chimera read is zero. I thought the -C option is a command used to not count chimeric reads ,  so why would adding the -C  command keep my  Unassigned_Chimera read count zero ?

Here is the script i used below 

#!/bin/bash

#PBS -N family_13_01_featureCount

#PBS -l nodes=2:ppn=8

#PBS -l vmem=32g

#PBS -l walltime=48:00:00 

#PBS -joe /hpf/projects/Leon/sequencing_repository/raw_fastq/RNA_tom/bam

#PBS -l mem=32g

set -euxo pipefail

cd /hpf/projects/Leon/sequencing_repository/raw_fastq/RNA_tom/bam

/hpf/tools/centos6/subread/1.5.3/bin/featureCounts -a /hpf/projects/Leon/sequencing_repository/raw_fastq/RNA_tom/bam/gencode.v19.annotation.gtf -o family_13_01_RNA-seq.counts -g gene_name -p -s 2 -C --donotsort  D4775_R312_BAligned.sortedByCoord.out.bam D4754_R311_BAligned.sortedByCoord.out.bam D4776_R310Aligned.sortedByCoord.out.bam D4777_R307Aligned.sortedByCoord.out.bam D4778_R308BAligned.sortedByCoord.out.bam D4828_R309_BAligned.sortedByCoord.out.bam 

[R adele]

I also have another question  I  used star 2.6.0  to do  my alignment and  I choose  the BAM SortedByCoordinate option . But I noticed  that you can also do  the BAM UnSorted option. Does it matter which option  you  choose,  will it affect  your read count, when using Featurecounts?

[Wei Shi]

No it wouldn't affect your counting.

[Wei Shi]

Could you try not to use '--donotsort' option to see if you can get counts for chimeric fragments? 

[R adele]

why do you suggest turning off the  '--donotsort' option  to get counts for chimeric fragments?

[Wei Shi]

Turning on '--donotsort' option could lead to incorrect counting results if your BAM/SAM reads are location sorted.