Combination Of Paired-End And Single-End Samples In Chip-Seq Tf Study

Question

[Nick]

I have 2 batches of chip-seq samples:

(A) One biological SE replicate

This batch, actually, consists of 4 SE Chip-seq samples - one treatment, one control, both have IP controls. All are SE sequenced at a lab A

(B) Two biological PE replicates

This batch consists of 8 PE Chip-Seq samples - 2 treatment, 2 controls, all have IP controls. All are PE sequenced at a lab B.

The idea is to establish differential binding between the treatment and the controls. I plan to use macs and diffbind but I am open to suggestions.

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My first specific question is how to treat the PE samples. I see 3 options:

(1) Treat all forward and reverse reads as SE reads

(2) Take just the forward or the reverse reads

(3) Treat each PE sample as, essentially, 2 technical replicates (one consisting of forward reads, one of reverse)

I find (3) intuitively attractive but, again, I am open to suggestions. If I go for it than the model would need to introduce 2 additional factors - one accounting for the technical replicates, another for the batch effect. So far I have dealt with batch effects only - I presume that adding additional factor shall be straight-forward but, please, let me know if there are things that I need to pay attention to.

Answer 1

[Istvan Albert]

There shouldn't be a need to alter them in any way. Having paired end data improves alignment but it does not alter your results.

more specifically

1. merge: you can do this if the majority of pairs overlap (>85%), this advice may be applied in general, simplifies life having only single long reads
2. no: you will lose half of the useful data
3. no: it is not a technical replicate, you would be treating a PE data as single end data and lose the connection between the pairs but you can't actually gain more information that is not already there

[Nick]

Thanks, but I meant not merging the reads themselves but, rather, merge, the two read files and treat the reads in the merged file as being all SE reads. The individual reads will stay as they are. Apologies for the confusion - will edit the question