Greetings,

I am interested in using the FourCSeq pipeline for the analysis of some Capture-C data I have generated in replicate.  One question I have is how to handle the situation where only one 4-bp cutter enzyme was applied (DpnII) and not two different enzymes like in standard 4C-seq applications.  Do I enter the  DpnII recognition sequence (GATC) twice for both reSequence1 and reSequence2? 

Since Capture-C uses 160 bp biotinylated probes for capture vs an inverse PCR with primers, I assume it is okay to enter my probe sequence (1 of a possible 2) that identifies my viewpoint fragment?

Are there any other items that I need to take into consideration when either setting up this pipeline or interpreting the results for Capture-C data?

Look forward to your responses,

Mark

Hi Mark,

entering the sequence twice might work. But you could also use another 4 cutter sequence. Reads will be counted at the ends of the first cutter used. The second cutter is used to define whether a fragment end is "valid" based on fragment size and whether there is a cutting site of the second cutter in the fragment.
So in your case you can set all fragments in the granges fragment object to valid once you created the fourc object. Then the analysis should correctly count your reads.

Best regards,
Felix