Question: counting reads with HOMER and only .bed files
0
gravatar for xAZx
4 months ago by
xAZx0
xAZx0 wrote:

Hi All,

I am brand new to ChIP seq and this type of analysis! Very excited to learn but unfortunately currently at a roadblock.

I have .BED files after peak calling with MACS, and unfortunately don't have access to any upstream alignment/BAM files. I have 62 files total, from 62 different samples. I have been trying to use HOMER to count reads, and eventually create a count matrix, but I'm stuck on making Tag directories. It states that it requires alignment files, which I don't have, so not sure how to proceed.

I also looked at using bedtools "multicov", but this looks like it requires BAM files as well. I can't seem to make any progress to figure out what to do next, so any help would be truly appreciated. Many thanks.

counting reads chip-seq homer • 188 views
ADD COMMENTlink written 4 months ago by xAZx0
2

MACS outputs peak regions, nothing more. If you want counts, you'll need the BAM files. No way around it. What is the ultimate goal?

ADD REPLYlink written 4 months ago by ATpoint21k

Thank you for your reply. The goal is to count reads on the merged peak set and gene body regions, finally get a count matrix with rows for each gene/peak and columns for each sample. And then determine differentially expressed peaks/genes by the DESeq2 package.

Do I still need all of the BAM files for the 62 samples to be able to do this?

ADD REPLYlink written 4 months ago by xAZx0

if you want to count reads, then you need a .bam file. Peaks files (.bed) do not contain reads.

ADD REPLYlink modified 4 months ago • written 4 months ago by steve2.2k

Please note the vocabulary (I do not want to educate you, please take no offence): A gene is expressed on the RNA level which can be measured by RNA-seq. ChIP-seq measures direct or indirect protein binding to the DNA. From this you can infer differential binding but not expression. ANyway, as I said, counts require BAMs, no way around it.

ADD REPLYlink written 4 months ago by ATpoint21k
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