[Bioc-devel] Additional summarizeOverlaps counting modes for ChIP-Seq

Ryan rct at thompsonclan.org
Thu May 1 23:05:44 CEST 2014

Hi Valerie,

On Thu May 1 13:27:16 2014, Valerie Obenchain wrote:
> I have some concerns about the *ExtraArgs() functions. Passing
> flexible args to findOverlaps in the existing mode functions
> fundamentally changes the documented behavior. The modes were created
> to capture specific overlap situations pertaining to gene features
> which are graphically depicted in the vignette. Changing 'maxgap' or
> 'minoverlap' will produce a variety of results inconsistent with past
> behavior and difficult to document (e.g., under what circumstances
> will IntersectionNotEmpty now register a hit).
Well, I wasn't so sure about those functions either. Obviously you can 
pass arguments that break things. They were mostly designed to be 
constructors for specific counting modes involving the minoverlap/maxgap 
arguments, but I decided I didn't need those modes after all. They're 
certainly not designed to be exposed to the user. I haven't carefully 
considered the interaction between the counting mode and 
maxgap/minoverlap, but I believe that it would be roughly equivalent to 
extending/shrinking the features/reads by the specified amount (with 
some differences for e.g. a feature/read smaller than 2*minoverlap). For 
example, with a read length of 100 and a minoverlap of 10 in Union 
counting mode, this would be the same as truncating the first and last 
10 (or mabe 9?) bases and operating in normal Union mode. As I said, 
though, there may be edge cases that I haven't thought of where 
unexpected things happen.
> I agree that controlling the overlap args is appealing and I like the
> added ability to resize. I've created a 'chipseq' mode that combines
> these ideas and gives what ResizeReads() was doing but now in 'mode'
> form. If this implementation gives you the flexibility you were
> looking for I'll check it into devel.

This sounds nice, but if I use the 'chipseq' mode, how do I specify 
whether I want Union, IntersectionNotEmpty, or IntersectionStrict? It 
looks like it just does Union? IntersectionStrict would be useful for 
specifying that the read has to occur entirely within the bounds of a 
called peak, for example. This is why I implemented it as a "wrapper" 
that takes another mode as an argument, so that the resizing logic and 
the counting logic were independent. Maybe summarizeOverlaps could 
accept an optional "read modification function", and if this is 
provided, it will pass the reads through this before passing them to the 
counting function. The read modification function would have to take any 
valid reads argument and return another valid reads argument. It could 
be used for modifying the reads as well as filtering them. This would 
allow resizing without the awkward nesting method that I've used.
> A couple of questions:
> - Do you want to handle paired-end reads? You coerce to a GRanges to
> resize but don't coerce back.
For paired end reads, there is no need to estimate the fragment length, 
because the pair gives you both ends of the fragment. So if I had 
paired-end ChIP-Seq data, I would use it as is with no resizing. I can't 
personally think of a reason to resize a paired-end fragment, but I 
don't know if others might need that.

I corece to GRanges because I know how GRanges work, but I'm not as 
familiar with GAlignments so I don't know how the resize function works 
on GAlignments and other classes. I'm sure you know better than I do how 
these work. If the coercion is superfluous, feel free to eliminate it.
> - Do you want to require strand info for all reads? Is this because of
> how resize() anchors "*" to 'start'?
Yes, I require strand info for all reads because the reads must be 
directionally extended, which requires strand info. Ditto for counting 
the 5-prime and 3-prime ends.

>> chipseq <- function(features, reads, ignore.strand=FALSE,
>> inter.feature=TRUE,
>> type="any", maxgap=0L, minoverlap=1L,
>> width=NULL, fix="start", use.names=TRUE)
>> {
>> reads <- as(reads, "GRanges")
>> if (any(strand(reads) == "*"))
>> stop("all reads must have strand")
>> if (!is.null(width))
>> reads <- do.call(resize(reads, width, fix=fix,
>> use.names=use.names,
>> ignore.strand=ignore.strand))
>> ov <- findOverlaps(features, reads, type=type,
>> ignore.strand=ignore.strand,
>> maxgap=maxgap, minoverlap=minoverlap)
>> if (inter.feature) {
>> ## Remove reads that overlap multiple features.
>> reads_to_keep <- which(countSubjectHits(ov) == 1L)
>> ov <- ov[subjectHits(ov) %in% reads_to_keep]
>> }
>> countQueryHits(ov)
>> }
> To count the overlaps of 5' and 3' ends:
> summarizeOverlaps(reads, features, chipseq, fix="start", width=1)
> summarizeOverlaps(reads, features, chipseq, fix="end", width=1)
> Valerie
> On 04/30/2014 02:41 PM, Ryan C. Thompson wrote:
>> No, I forgot to attach the file. Here is the link:
>> https://www.dropbox.com/s/7qghtksl3mbvlsl/counting-modes.R
>> On Wed 30 Apr 2014 02:18:28 PM PDT, Valerie Obenchain wrote:
>>> Hi Ryan,
>>> These sound like great contributions. I didn't get an attachment - did
>>> you send one?
>>> Thanks.
>>> Valerie
>>> On 04/30/2014 01:06 PM, Ryan C. Thompson wrote:
>>>> Hi all,
>>>> I recently asked about ways to do non-standard read counting in
>>>> summarizeOverlaps, and Martin Morgan directed me toward writing a
>>>> custom
>>>> function to pass as the "mode" parameter. I have now written the
>>>> custom
>>>> modes that I require for counting my ChIP-Seq reads, and I figured I
>>>> would contribute them back in case there was interest in merging them.
>>>> The main three functions are "ResizeReads", "FivePrimeEnd", and
>>>> "ThreePrimeEnd". The first allows you to directionally extend or
>>>> shorten
>>>> each read to the effective fragment length for the purpose of
>>>> determining overlaps. For example, if each read represents the 5-prime
>>>> end of a 150-bp fragment and you want to count these fragments
>>>> using the
>>>> Union mode, you could do:
>>>> summarizeOverlaps(mode=ResizeReads(mode=Union, width=150,
>>>> fix="start"), ...)
>>>> Note that ResizeReads takes a mode argument. It returns a function
>>>> (with
>>>> a closure storing the passed arguments) that performs the resizing (by
>>>> coercing reads to GRanges and calling "resize") and then dispatches to
>>>> the provided mode. (It probably needs to add a call to "match.fun"
>>>> somewhere.)
>>>> The other two functions are designed to count overlaps of only the
>>>> read
>>>> ends. They are implemented internally using "ResizeReads" with
>>>> width=1.
>>>> The other three counting modes (the "*ExtraArgs" functions) are
>>>> meant to
>>>> be used to easily construct new counting modes. Each function takes
>>>> any
>>>> number of arguments and returns a counting mode that works like the
>>>> standard one of the same name, except that those arguments are
>>>> passed as
>>>> extra args to "findOverlaps". For example, you could do Union mode
>>>> with
>>>> a requirement for a minimum overlap of 10:
>>>> summarizeOverlaps(mode=UnionExtraArgs(minoverlap=10), ...)
>>>> Note that these can be combined or "nested". For instance, you might
>>>> want a fragment length of 150 and a min overlap of 10:
>>>> myCountingMode <- ResizeReads(mode=UnionExtraArgs(minoverlap=10),
>>>> width=150, fix="start")
>>>> summarizeOverlaps(mode=myCountingMode, ...)
>>>> Anyway, if you think any of these are worthy of inclusion for
>>>> BioConductor, feel free to add them in. I'm not so sure about the
>>>> "nesting" idea, though. Functions that return functions (with states
>>>> saved in closures, which are then passed into another function) are
>>>> confusing for people who are not programmers by trade. Maybe
>>>> summarizeOverlaps should just gain an argument to pass args to
>>>> findOverlaps.
>>>> -Ryan Thompson
>>>> _______________________________________________
>>>> Bioc-devel at r-project.org mailing list
>>>> https://stat.ethz.ch/mailman/listinfo/bioc-devel

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