Question: counting reads with HOMER and only .bed files
gravatar for xAZx
15 months ago by
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 • 438 views
ADD COMMENTlink written 15 months ago by xAZx0

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 15 months ago by ATpoint36k

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 15 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 15 months ago • written 15 months ago by steve2.6k

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 15 months ago by ATpoint36k
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