Thanks Mike, that is what I thought. What if we wanted to perform kruskal wallis, or is it possible to perform anova on the variance-stabilized matrix? On Wed, Apr 16, 2014 at 2:29 PM, Michael Love wrote: > 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 > wrote: > > Please help with this? Thanks again. > > > > > > On Mon, Apr 14, 2014 at 6:02 PM, Sophie Josephine Weiss > > 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 > >> 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 > >>> 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 > >>> > > >>> > wrote: > >>> >> > >>> >> hi Sophie, > >>> >> > >>> >> > >>> >> On Mon, Apr 14, 2014 at 1:15 PM, Sophie Josephine Weiss > >>> >> 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 > >>> >> > 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 > >>> >> >> 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 > >>> >> >>> 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 > >>> >> >>>> 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 > >>> >> >>>>> 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 > >>> >> >>>>>> 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@r-project.org > >>> >> >>>>>>> https://stat.ethz.ch/mailman/listinfo/bioconductor > >>> >> >>>>>>> Search the archives: > >>> >> >>>>>>> > >>> >> >>>>>>> > http://news.gmane.org/gmane.science.biology.informatics.conductor > >>> >> >>>>>> > >>> >> >>>>>> > >>> >> >>>>> > >>> >> >>>> > >>> >> >>> > >>> >> >> > >>> >> > > >>> > > >>> > > >> > >> > > > [[alternative HTML version deleted]]