featurecounts -p Paried ends which count total to use as read1 &read 2 give different totals.

Question

[ray.liv.wong]

Hi

First post so please be kind, its my first year.

I have run featurecounts -p on my files and the output has a count total for read 1 and read 2. My simple question is which total count should i use? read 1 or read 2 since they are not the same.

I tried finding the answer on the forumn but no luck.

Thanks for your help

R

[Carlo Yague]

featurecounts -p counts fragments if the reads are paired, I have never seen it reporting separated counts for R1 and R2... What is the exact command you use ? And can you show the summary results ?

[ray.liv.wong]

To generate Bam file i used hisat2

hisat2 -x "./refs/hg38_hisat2_index/hg38_tran/genome_tran" -1 "${file1}" -2 "${file2}" -S "./PRJNA316673_sam/${acc}.sam"
samtools sort -l 102 -o "./PRJNA316673_bam/${acc}.bam" "./PRJNA316673_sam/${acc}.sam"

Then for quantification

ref="./refs/hg38_ensembl_gtf/ensembl_gtf/hg38.gtf.gz"
list=(./PRJNA316673_bam/*)

featureCounts -p -T 16 -t exon -g gene_id -a $ref --tmpDir "./temp" -o "./PRJNA316673_3_counts.txt" "${list[@]}"

output is like this

Geneid          Chr                   Strand            Length  read1   read2 
ENSG00000223972 1;1;1;1;1;1;1;1;1     +;+;+;+;+;+;+;+;+ 1735    0         0 
ENSG00000227232 1;1;1;1;1;1;1;1;1;1;1   ;-;-;-;-;-;-;-;-;-;-;-  1351    42        49    
ENSG00000268903         1                                -                           755             50        33

[Carlo Yague]

what is the output of ls PRJNA316673_bam/*?

[Rogerio Ribeiro]

Can you post the full command you used? Also, can you post the command used to generate your alignment (i.e bam) file?