Dear Allen,
it is certainly possible to modify the getGRange() according to your
specific needs, please see the description of the scanBamFlag
constructor
in the Rsamtools manual. The getGRange() function is part of the file
MEDIPS.readRegionsFile.R. However, I do not know how straight forward
it
will be to source and override functions in a session. In case you
intend
to ignore multiple mappers in your downstream analysis, you might want
to
suppress them in the output of the alignment tool and use MEDIPS as it
is.
Stacked reads might be artifacts due to PCR amplification, but they
can
also occur in case of over-sequencing. Ambitious PCR amplification
might
result in an enrichment of reads at exactly the same position not
related
to an enrichment of the actual mark of interest (e.g. mC).
All the best,
Lukas
On Wed, Aug 27, 2014 at 10:07 AM, Chong Kim San Allen <patcksa at="" nus.edu.sg="">
wrote:
> Dear Lukas and Wade.
> Thanks for your replies. It is very much appreciated.
>
> Lukas, you mentioned that the internal MEDIPS function getGRange()
is set
> so as to only ignore all unmapped reads and this is done through
> scanBamFlag. I was wondering if it is possible for me to modify the
> getGRange() function so as to only use unique single-mapped reads or
do I
> really need to filter my bam file as Wade suggested.
>
> Wade, thanks for highlighting "uniq=TRUE" for duplicate/stacked
reads. I
> actually had not considered this would be problem because (correct
me if I
> am wrong) I thought that the number of reads for a peak was an
indicator of
> the degree of methylation, much like the quantitation of methylation
given
> by beta or M values for arrays. So, my question is why would one
want to
> remove such stacked reads?
>
> I look forward to hearing to your answers and thank you for your
time and
> assistance.
>
> best,
> Allen
> ________________________________________
> From: Lukas Chavez [lukas.chavez.mailings at googlemail.com]
> Sent: Wednesday, August 27, 2014 3:50 AM
> To: Davis, Wade
> Cc: Allen [guest]; bioconductor at r-project.org; Chong Kim San
Allen;
> MEDIPS Maintainer
> Subject: Re: [BioC] How does MEDIPS handle multiple mapping of a
read
>
> Dear Allen and Wade,
>
> indeed "multiple mappers" and reads that align to exactly the same
> position (sometimes named "clonal" or "stacked" reads) are two
different
> things and need to be discussed separately. Wade has given a great
overview
> about "stacked reads" by referring to the uniq=TRUE parameter of the
MEDIPS
> package, sharing his results with the IDR, and by pointing to a
study that
> further discusses this topic. Here, I would like to add that some
tools
> provide the opportunity to estimate a global or local maximum of
"stacked"
> reads. MEDIPS currently only allows for considering one
representative per
> genomic position and strand or all reads. It is somewhere on my ToDo
list
> to add a third option allowing a certain maximum of stacked reads.
>
> Reads that align to several positions in the genome ("multiple
mapper")
> are common in MeDIP experiments, because of high abundance of
methylation
> at repetitive regions. MEDIPS uses functions of the Rsamtools
package to
> import mapping results from a bam file. Personally, I do not have
> experience using bam files including multiple alignments per read,
because
> I am typically reporting only mapping results for unique mappers.
However,
> if a bam file contains multiple mappers, all hits will be imported
into
> MEDIPS. This behavior is determined by the internal MEDIPS function
> getGRange(). The relevant line is
>
> scanFlag = scanBamFlag(isUnmappedQuery = F)
>
> Here, all unmapped reads are neglected in the scanBamFlag
constructor.
> Among others, the scanBamFlag constructor has the parameter
> isNotPrimaryRead which is NA by default and the parameter is not
changed by
> MEDIPS. Consequently, all alignments are returned regardless of
their
> primary status. More information on the primary status of an
alignment (
>
http://www.bioconductor.org/packages/release/bioc/manuals/Rsamtools/
man/Rsamtools.pdf
> ):
>
> As far as I remember, I previously encountered problems when
importing
> CSEM alignment results into MEDIPS, but this is a while ago. What
you can
> always do is to convert your mapping results/ bam file into a plain
bed
> file, regardless of the presence of multiple alignments per read and
> regardless of the appropriate use of flags of any alignment tool.
MEDIPS
> will then consider each given entry as a mapping hit and calculate
the
> genomic coverage at genome wide windows accordingly.
>
> Best regards,
> Lukas
>
>
>
> On Tue, Aug 26, 2014 at 9:28 AM, Davis, Wade <davisjwa at="" health.missouri.edu=""> <mailto:davisjwa at="" health.missouri.edu="">> wrote:
> Hi Allen,
> It seems there are two somewhat related issues that are relevant
here:
> multi-mapped reads (i.e., NOT uniquely MAPPABLE read) and duplicate
reads
> (i.e., not the only read with a given same start/stop). I like to
avoid the
> term "unique" by itself because it can be ambiguous as you can see.
>
> To my knowledge, MEDIPS doesn't address the multi-mapped reads; I
think
> many would argue that is something best handled by the aligner or
afterward
> by filtering your bam files based on certain flags. Specifically, it
is
> more efficient for the aligner to implement whatever you desire
(e.g.,
> spreading them around uniformly or based on some estimated
probability)
> during alignment than for MEDIPS to do it.
>
> However, MEDIPS does deal with duplicate reads via the uniq flag, as
noted
> in the vignette:
> "MEDIPS will replace all reads which map to exactly the same start
and end
> positions on the same strand by only one representative:
> > uniq=TRUE"
>
> See ?MEDIPS.createSet
>
> Most of what I have read about for ChiP/MeDIP lead us to only count
> duplicate reads once (i.e., use uniq=TRUE). We looked at a number of
> samples analyzed both ways and looked at the Irreproducible
Discovery Rate
> (IDR) plots and found better reproducibility using this approach.
Another
> paper suggests using the duplicates or discarding them depending on
the
> purpose:
>
>
http://www.ploscompbiol.org/article/info%3Adoi%2F10.1371%2Fjournal.p
cbi.1003326
>
> I hope this helps a little,
> Wade
>
> -----Original Message-----
> From: Allen [guest] [mailto:guest at bioconductor.org<mailto:> guest at bioconductor.org>]
> Sent: Tuesday, August 26, 2014 4:50 AM
> To: bioconductor at r-project.org<mailto:bioconductor at="" r-project.org="">;
> patcksa at nus.edu.sg<mailto:patcksa at="" nus.edu.sg="">
> Cc: MEDIPS Maintainer
> Subject: [BioC] How does MEDIPS handle multiple mapping of a read
>
> Hi,
> I was wondering if someone can tell me what happens in MEDIPS a
particular
> read maps to multiple locations on the genome? I have just aligned
my
> MeDIPS-seq data using BWA and only about 40% have a mapping quality
of
> above 30. Does MEDIPS only take into account unique reads and throws
out
> the rest?
>
> I looked at the MEDIPS manual (
>
http://www.bioconductor.org/packages/release/bioc/vignettes/MEDIPS/i
nst/doc/MEDIPS.pdf)
> and can't find any information on this.
>
> I read on various forums and many people suggest just using unique
reads.
> However, for ChIP-seq, it is suggested that one could use CSEM so
that
> information from multiple mapping is not completely lost.
>
> Thanks in advance for your help.
>
> Allen
>
>
>
> -- output of sessionInfo():
>
> error
>
> --
> Sent via the guest posting facility at bioconductor.org<
>
http://bioconductor.org>.
>
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