How to create SAF from text file for FeatureCounts

Question

[pt.taklifi]

Hello everyone,

I have some ATACseq bam files and a reference peak set(genomic regions) in text format. I am trying to get count matrix from the bam files( i.e or each bam file I want to calculate f how many reads fall in each peak) . so I am trying to use featureCounts for this purpose. which needs annotations in SAF or GTF format. 1) in my case is annotation the reference peakset ? 2) if so how to convert my peak set to SAF or GTF format.

Here are a few lines of the peak set

seqnames    start   end name    score   annotation  percentGC
chr1    906012  906513  ACC_10  7.171192997 Intron  0.612774451
chr2    112541661   112542162   ACC_10008   22.03057903 Promoter    0.55489022
chr1    21673421    21673922    ACC_1001    6.459954383 Distal  0.508982036
chr2    112584205   112584706   ACC_10013   43.20855549 Promoter    0.586826347
chr2    112596243   112596744   ACC_10016   5.428209077 Intron  0.491017964
chr1    21725692    21726193    ACC_1002    5.201272875 Intron  0.405189621

Answer 1

[ATpoint]

Converting from BED to SAF/GFF

If your file has a header you will need to skip the first line, in awk that would be via NR>1. You can convert this file you have there to SAF and then basically do something like:

featureCounts -a your.saf -F SAF -o counts.txt *.bam

[pt.taklifi]

thank you very much.

[pt.taklifi]

one following question: my bam files are not in the same directory, so I created a text file containing all bam file locations like :acc_bamFiles.txt here is a few lines:

SRR10984460/bam/SRR10984460.dedup.bam
SRR10984461/bam/SRR10984461.dedup.bam
SRR10984462/bam/SRR10984462.dedup.bam

however when I try :

featureCounts -a PanCancer_PeakSet.saf -F SAF -o counts.txt acc_bamFiles.txt

I get this error message:

ERROR: invalid parameter: 'acc_bamFiles.txt'

so should I just run featureCounts for each bam file separately and then append count matrixes together or is there an easier way ?

[ATpoint]

I do not think that this can be a text file. I usually make symbolic links to the directory where the SAF file is. Say you are in the SAF directory use ln -s /path/to/bam . for all BAMs and then use *.bam.

[pt.taklifi]

thank you very much!