[BioC] phyloseq/DESeq gives negative transformed values

[Michael Love]

hi Sophie,

We recommend using the standard DESeq() function for differential expression.

This is mentioned in the first line of the vignette section on transformations:

"In order to test for diff erential expression, we operate on raw
counts and use discrete distributions as
described in the previous section"

Also, in the McMurdie and Holmes, they are using the DESeq() function,
as shown in their supplemental material:

http://joey711.github.io/waste-not-supplemental/simulation-differential-abundance/simulation-differential-abundance-server.html

On Wed, Apr 16, 2014 at 3:22 PM, Sophie Josephine Weiss
<Sophie.Weiss at colorado.edu> wrote:
> Please help with this?  Thanks again.
>
>
> On Mon, Apr 14, 2014 at 6:02 PM, Sophie Josephine Weiss
> <Sophie.Weiss at colorado.edu> wrote:
>>
>> Thanks again Mike - would it be ok to do chi-2 and other significance
>> tests on the DESeq transformed datasets using independent code, or is it
>> necessary to do the differential expression tests strictly within DESeq2?
>>
>> Sophie
>>
>>
>> On Mon, Apr 14, 2014 at 5:41 PM, Michael Love
>> <michaelisaiahlove at gmail.com> wrote:
>>>
>>> hi Sophie,
>>>
>>> The VST code is the same in DESeq and DESeq2. The estimation of
>>> dispersion is slightly different (details are in the vignette "Changes
>>> from DESeq to DESeq2"), but the fitted line (which is used by the VST)
>>> should be very similar.
>>>
>>> Mike
>>>
>>> On Mon, Apr 14, 2014 at 6:27 PM, Sophie Josephine Weiss
>>> <Sophie.Weiss at colorado.edu> wrote:
>>> > Hi Mike,
>>> > The McMurdie and Holmes paper uses DESeq for matrix normalization - do
>>> > you
>>> > think that is ok, or would it be better to use DESeq 2?
>>> > Thanks again,
>>> > Sophie
>>> >
>>> >
>>> > On Mon, Apr 14, 2014 at 3:40 PM, Michael Love
>>> > <michaelisaiahlove at gmail.com>
>>> > wrote:
>>> >>
>>> >> hi Sophie,
>>> >>
>>> >>
>>> >> On Mon, Apr 14, 2014 at 1:15 PM, Sophie Josephine Weiss
>>> >> <Sophie.Weiss at colorado.edu> wrote:
>>> >> >
>>> >> > Hi Mike,
>>> >> > Thanks for the references.  By "threshold at 0" do you mean set any
>>> >> > negative values equal to 0?
>>> >>
>>> >>
>>> >> yes.
>>> >>
>>> >>
>>> >> >
>>> >> > Do you think this is the best approach?
>>> >>
>>> >>
>>> >> I haven't explored this area, and would defer to the McMurdie and
>>> >> Holmes paper for the best combinations of distance and transformation.
>>> >>
>>> >>
>>> >> >
>>> >> > Thanks again,
>>> >> > Sophie
>>> >> >
>>> >> >
>>> >> > On Mon, Apr 14, 2014 at 11:01 AM, Michael Love
>>> >> > <michaelisaiahlove at gmail.com> wrote:
>>> >> >>
>>> >> >> I tried poking around here
>>> >> >> http://joey711.github.io/phyloseq/distance
>>> >> >> but couldn't see if the authors did anything for distances
>>> >> >> requiring
>>> >> >> non-negative data. It appears
>>> >> >>
>>> >> >> http://www.ploscompbiol.org/article/info%3Adoi%2F10.1371%2Fjournal.pcbi.1003531
>>> >> >> that VST was tested with Bray-Curtis distance. I think the distance
>>> >> >> is
>>> >> >> designed for counts, but you could always threshold at 0 to insist
>>> >> >> that the
>>> >> >> log2-like quantity act more like a count.
>>> >> >>
>>> >> >>
>>> >> >>
>>> >> >> On Mon, Apr 14, 2014 at 12:23 PM, Sophie Josephine Weiss
>>> >> >> <Sophie.Weiss at colorado.edu> wrote:
>>> >> >>>
>>> >> >>> Hi Mike,
>>> >> >>> Thanks for explaining more.  I am used to working with rarefied
>>> >> >>> microbial datasets, that is why.  Instead of rarefying I would
>>> >> >>> like to use
>>> >> >>> the DESeq method.
>>> >> >>>
>>> >> >>> How would you then suggest going about calculating bray-curtis
>>> >> >>> distance, or summarized taxa diagrams with these new transformed
>>> >> >>> matrices
>>> >> >>> with negative values?
>>> >> >>> Thanks again,
>>> >> >>> Sophie
>>> >> >>>
>>> >> >>>
>>> >> >>> On Mon, Apr 14, 2014 at 7:17 AM, Michael Love
>>> >> >>> <michaelisaiahlove at gmail.com> wrote:
>>> >> >>>>
>>> >> >>>> hi Sophie,
>>> >> >>>>
>>> >> >>>> Can you explain why you don't want negative values in the
>>> >> >>>> transformed
>>> >> >>>> values?  Adding one to the raw counts is not sufficient. I should
>>> >> >>>> have said
>>> >> >>>> in my previous email, "the expected counts on the common scale".
>>> >> >>>> If the
>>> >> >>>> size factor for a sample is 2, then an expected count of 1 leads
>>> >> >>>> to an
>>> >> >>>> expected count of 1/2 on the common scale (after accounting for
>>> >> >>>> size
>>> >> >>>> factors).
>>> >> >>>>
>>> >> >>>>
>>> >> >>>> On Sun, Apr 13, 2014 at 11:50 PM, Sophie Josephine Weiss
>>> >> >>>> <Sophie.Weiss at colorado.edu> wrote:
>>> >> >>>>>
>>> >> >>>>> Hi Mike,
>>> >> >>>>> Thanks for your reply!  Ok, makes sense, but I added 1 to all my
>>> >> >>>>> matrix values, so the lowest value in the matrix is 1 - there
>>> >> >>>>> are still
>>> >> >>>>> negatives?
>>> >> >>>>> Thanks again,
>>> >> >>>>> Sophie
>>> >> >>>>>
>>> >> >>>>>
>>> >> >>>>> On Sun, Apr 13, 2014 at 9:01 PM, Michael Love
>>> >> >>>>> <michaelisaiahlove at gmail.com> wrote:
>>> >> >>>>>>
>>> >> >>>>>> hi Sophie,
>>> >> >>>>>>
>>> >> >>>>>> The transformations in DESeq and DESeq2 are log2-like
>>> >> >>>>>> transformations. If the expected count is between 0 and 1, the
>>> >> >>>>>> values can be
>>> >> >>>>>> negative, this does not indicate a problem.
>>> >> >>>>>>
>>> >> >>>>>> Mike
>>> >> >>>>>>
>>> >> >>>>>>
>>> >> >>>>>> On Sun, Apr 13, 2014 at 5:17 PM, Sophie Josephine Weiss
>>> >> >>>>>> <Sophie.Weiss at colorado.edu> wrote:
>>> >> >>>>>>>
>>> >> >>>>>>> Hello,
>>> >> >>>>>>> I have microbiome data with no replicates, from different
>>> >> >>>>>>> conditions.  I am
>>> >> >>>>>>> trying to transform the data using the DESeq method, as
>>> >> >>>>>>> described
>>> >> >>>>>>> in
>>> >> >>>>>>> McMurdie and Holmes 2014.
>>> >> >>>>>>>
>>> >> >>>>>>> The attached file is the definition I am using, as per the
>>> >> >>>>>>> supplemental
>>> >> >>>>>>> info in McMurdie and Holmes 2014, and the .biom file I am
>>> >> >>>>>>> using.
>>> >> >>>>>>>
>>> >> >>>>>>> Thank you for your help,
>>> >> >>>>>>> Sophie
>>> >> >>>>>>>
>>> >> >>>>>>> _______________________________________________
>>> >> >>>>>>> Bioconductor mailing list
>>> >> >>>>>>> Bioconductor at r-project.org
>>> >> >>>>>>> https://stat.ethz.ch/mailman/listinfo/bioconductor
>>> >> >>>>>>> Search the archives:
>>> >> >>>>>>>
>>> >> >>>>>>> http://news.gmane.org/gmane.science.biology.informatics.conductor
>>> >> >>>>>>
>>> >> >>>>>>
>>> >> >>>>>
>>> >> >>>>
>>> >> >>>
>>> >> >>
>>> >> >
>>> >
>>> >
>>
>>
>