How to Intersect Replicates in ChIPseq workflow

Question: How to Intersect Replicates in ChIPseq workflow

20 months ago by

Hello, I have some ChIPseq data but I do not know how to write/use code so I am trying to build a workflow on UseGalaxy.org to analyze Treatment-vs-Control in 3 biological replicates sequenced on Illumina. Based on some of the online forums, I need to "intersect" (and not "merge") the data between 3 biological replicates.

1) On pipeline design: - Do I need to fist run the intersct of 3 dataset (e.g. 3 treatments, 3 control), then run the output of 3 treatment against the output of 3 controls on MACS. - or, do I need to first run MACS on Treatment vs Control for each biological replicate, then run intersect on the output files.

2) On the use of the Multiple Intersect function (under BEDTools) in building a Workflow: How to I compare 2 or 3 files if the total allowed input is 1 BED file and 1 genome file? Am I misunderstanding what the purpose of the tool is?

Any expert opinion would be greatly appreciated! Dan

worfflow macs chipseq intersect bedtools • 609 views

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modified 20 months ago • written 20 months ago by georgess.dan • 40

20 months ago by

Mo Heydarian ♦ 830

United States

Mo Heydarian ♦ 830 wrote:

Hello,

Have a look at this tutorial on chIP-seq analysis, it may help: https://galaxyproject.github.io/training-material//ChIP-Seq/tutorials/TAL1_binding_site_identification

You will want to intersect your peaks after running the aligned data through MACS.

In the workflow editor you can link multiple input files to the same node for the multiple intersect function of BEDtools.

Great questions! Hope this helps!

Cheers,

Mo Heydarian

ADD COMMENT • link written 20 months ago by Mo Heydarian ♦ 830

20 months ago by

georgess.dan • 40

georgess.dan • 40 wrote:

Hi Mo, Thanks for tutorial. It looks super helpful. I'm not sure how to create a multi-file node. The Multiple Intersect function in BEDTools only has 2 input slots (1 for "Genome file" and 1 for BED file) and allows for one input file per slot.

Thanks, Dan

ADD COMMENT • link written 20 months ago by georgess.dan • 40

Hi Dan, You are correct, you can't add multiple inputs to the input node of Multiple Intersect. Sorry for the confusion.

In the workflow editor on the tool form of Multiple Intersect, try changing the first parameter (Sample name) from "Use input's tag" to "Enter custom name per file". This will enable more input slots for the files you want to intersect (and you can add more if you desire).

Hope this helps!

Cheers, Mo Heydarian

ADD REPLY • link written 20 months ago by Mo Heydarian ♦ 830

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