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

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.

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.

-Ryan

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