Question: I am updating the SICER algorithm ( https://github.com/endrebak/epic ) and have added paired end support. The original SICER does not support paired end reads, but for the single end case it reduces each read to a single coordinate, which is the start of the read plus half the fragment size.

I have no paired end ChIP-Seq data myself to try it on, so I might have implemented a too naive paired end mode. It reduces each paired end read to a point by taking the leftmost and rightmost coordinate of (both the starts and ends of) a pair and finding its midpoint.

So for the (fake) read pair

chr7	20246668	20246669	chr7	20246693	20246694	U0	0	+	+

the coordinate is

20246694 + (20246694-20246668)/2 = 20 246 707

It seems like this might lead to a problem though:

If the two mates are very far apart, the midpoint might be in a bad (ie heterochromatic) or uninteresting (ie blacklisted) region.

What is the best way to solve this?

I can think of two solutions, but do not understand all their up- and downsides:

1) Discard read pairs more than say 100 bp apart
2) Treat each mate in a pair as an individual read

I lean towards the first solution since it seems like the paired end libraries my users use contains much much more data than a typical single end library, and doubling that amount seems like it would be a lot of pain (waiting) for little gain (better results).

Answer: What a well-timed and IMHO interesting post :-)

I am working on adding paired end (PE) support to [EaSeq][1], and I am going through exactly the same considerations and do not have any prior experience with PE as we have not used it for ChIP at our institute (yet). EaSeq also works with a single integer coordinate internally to allow many data sets to be loaded in memory simultaneously.

Back to your question: Midpoint sounds best to me, but I am not entirely sure why you would like to filter the reads as you propose. As I understand it then some people actually use PE ChIP-seq to map some reads to areas that are normally hard to map (e.g. heterochromatic regions). So at first thought I would leave those within the data set, but I would be very happy to learn from other opinions on this.

Moreover, for many ChIP-seq experiments you would probably discard a large fraction of the reads by setting the cutoff at 100 bp. I would like to know when/why you would prefer keeping/discarding these reads - in many cases additional (but larger) pairs would give stronger statistics, but reduce resolution. However, the question is whether this is a real-life issue for most applications? From my own experience the app. 20 bp resolution that I typically get from my peakfinding algorithm (see e.g. [figure 5C here][2]) is sufficient to get really nice de novo motif discovery with MEME and good central motif distribution with Centrimo - even when working on bulk single read ChIP-seq with an experimentally determined main fragment lenght of >100 bp.

Regarding the blacklisted regions, why not just remove read pairs that overlap with these in the upstream process? Although, I can also see that this might create a strange 'event horizon' in the vicinity of blacklisted regions as an increasing fraction of the pairs are discarded. It might be preferable to keep peak-finding simple and filter peaks from blacklisted regions later on?

For EaSeq, I think that the best solution would be to rewrite the central part of the code to accomodate two (or four) coordinates when that is prefered over an efficient memory footprint. That would 1) eliminate the annoying rounding error for odd sized fragments (and potentially a 1 bp shift when exporting data again) as well as allow for downstream visualization and filtering of reads based on distance between the pairs.

[1]: https://www.biostars.org/p/185271/
[2]: http://www.nature.com/nsmb/journal/v23/n4/full/nsmb.3180.html