Doubt using HOMER annotatePeaks.pl

Question

[nanoide]

Hi all, I've tried to annotate MACS2 ChIP-seq peaks using HOMER annotatePeaks.pl. I have just given to annotatePeaks.pl the annotation and the fasta files for my genome and it's working.

I'm not sure however about the column "Distance to TSS" on the annotation. The features I've given in the annotation are CDS, exon, 5' UTR, 3' UTR, gene, start codon, stop codon and transcript. I don't have TSSs specifically annotated. What's the reference HOMER is using for giving a number (and a sign, upstream or dowstream) in the "Distance to TSS" column? It migth be the ATG? I have failed to find this info on the documentation. And the distance would be from the middle of the peaks or the start/ending?

Hope anyone have any idea.

Thanks for your time.

Answer 1

[Benn]

Homer's annotatePeaks.pl is using RefSeq TSS annotation by default, except when using the option "-cTSS <peak/pos file>". Did you use that option? What command did you use?

It is explained here.

If you want to know how the distance is calculated, try to find out from your data, check for the coordinate of the TSS in the .tss file, and see how it relates to your peak coordinates. Do the maths!

[nanoide]

Yes, I'm sorry. Should have explained myself better

I'm using a custom genome without annotated TSSs. I have used: annotatePeaks.pl bed_file fasta_file -gtf annotation_gtf > txt_file

annotation_gtf contains coordinates for different features: CDS, exon, 5' UTR, 3' UTR, gene, start codon, stop codon and transcript There is no RefSeq TSS for this organisms, no "TSS" in the features I have provided and I have not used -cTSS. I have also read about the command -ann and creating a custom table annotation for homer, but for now this is working and it seems to make sense. I'm just wondering what exactly is HOMER using in this case as "TSS"

Thanks!

[bencom]

Hi,

I am trying to use HOMER with ensembl gtf with a command like :

annotatePeaks.pl bed_file -gtf annotation_gtf > txt_file

When I do that I don't get the UTRs in the output.

Do you know if it's the fasta file that make them happen in the output file ?

Do you have any other suggestion to solve this issue ?

Thank you ! Ben