Hello All: I am Yogender Gowtham, a graduate student from Clemson University. We are using RNA-Seq to investigate the transcriptional changes of Chinese Hamster Ovary (CHO) cells during serum-free adaptation. Our experiment has two cell lines, say Y and Z, under 3 different serum concentrations, say, 10%, 5% and 0%. Cell line Z could not be adapted to 0% serum condition and hence we have unbalanced set of conditions. For clarity, the conditions are listed below A - Y at 0% D - Z at 5% B - Y at 5% E - Z at 10% C - Y at 10% We have come up with 9 different pairwise comparisons based on the scientific questions we want to answer. But performing so many pairwise comparisons will lead to more liberal FDR values. Hence we want to perform ANOVA analysis on all the five conditions. Based on section 3.2.6 which explains how to perform ANOVA-like analysis. >From what I understand, it contrasts each column with intercept and will list any differences that are non-zero (B-A, C-A, D-A, E-A). But ideally we want B-A, B-C, B-D, E-B, C-A, D-C, E-C, D-A, or E-A comparisons in an ANOVA test. If anyone can suggest a method on edgeR to perform ANOVA tests, that will be perfect. If not, can someone suggest other possible packages that could help me do an ANOVA analysis on multiple groups. Regards Yogender Kumar Gowtham Graduate Research Assistant Clemson University Department of Bioengineering 202 Biosystems Research Complex Clemson, SC (864)650-7451 [[alternative HTML version deleted]]

I khy Sent from my iPhone > On Aug 1, 2014, at 12:52 AM, Yogender Gowtham <ygowtha at="" g.clemson.edu=""> wrote: > > Hello All: > > I am Yogender Gowtham, a graduate student from Clemson University. We are > using RNA-Seq to investigate the transcriptional changes of Chinese Hamster > Ovary (CHO) cells during serum-free adaptation. Our experiment has two cell > lines, say Y and Z, under 3 different serum concentrations, say, 10%, 5% > and 0%. Cell line Z could not be adapted to 0% serum condition and hence we > have unbalanced set of conditions. For clarity, the conditions are listed > below > > A - Y at 0% D - Z at 5% > B - Y at 5% E - Z at 10% > C - Y at 10% > > We have come up with 9 different pairwise comparisons based on the > scientific questions we want to answer. But performing so many pairwise > comparisons will lead to more liberal FDR values. Hence we want to perform > ANOVA analysis on all the five conditions. > > Based on section 3.2.6 which explains how to perform ANOVA-like analysis. >> From what I understand, it contrasts each column with intercept and will > list any differences that are non-zero (B-A, C-A, D-A, E-A). But ideally we > want B-A, B-C, B-D, E-B, C-A, D-C, E-C, D-A, or E-A comparisons in an ANOVA > test. > > If anyone can suggest a method on edgeR to perform ANOVA tests, that will > be perfect. If not, can someone suggest other possible packages that could > help me do an ANOVA analysis on multiple groups. > > Regards > > > Yogender Kumar Gowtham > Graduate Research Assistant > Clemson University > Department of Bioengineering > 202 Biosystems Research Complex > Clemson, SC > (864)650-7451 > > [[alternative HTML version deleted]] > > _______________________________________________ > 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

Dear Yogender Gowtham, The edgeR user guide tells you how to do the anova test, as you have already noted for yourself. It really is as simple as the user's guide says. What you are not understanding is that if B-A, C-A, D-A and E-A are all zero, then it follows that the gene must have the same expression level in all 5 conditions, and hence all the other possible comparisons (B-C, D-E etc etc) must be zero as well. In fact any four contrasts between the five conditions are sufficient. If any four contrasts are zero, then all must be zero. This is why the anova test for five conditions is on 4 degrees of freedom. edgeR will return you the same correct F test whichever set of 4 contrasts you ask it to test. This means you will get the same test results from testing B-A, C-A, D-A and E-A simultaneously as you would from E-D, D-C, C-B and B-A, or indeed from any other set of four independent contrasts. By the way, it is not true that performing pairwise t-tests will necessarily give liberal FDR values. If the FDR is controlled correctly for each individual comparison, then it is automatically controlled for the whole set of tests. You may be thinking of p-values and type I error rates, but FDRs scale up differently to type I error rates. There is actually little to be gained in the genomic context from doing an anova test if you are going to follow it up by posthoc t-tests. Best wishes Gordon > On Aug 1, 2014, at 12:52 AM, Yogender Gowtham <ygowtha at="" g.clemson.edu=""> wrote: > > Hello All: > > I am Yogender Gowtham, a graduate student from Clemson University. We > are using RNA-Seq to investigate the transcriptional changes of Chinese > Hamster Ovary (CHO) cells during serum-free adaptation. Our experiment > has two cell lines, say Y and Z, under 3 different serum concentrations, > say, 10%, 5% and 0%. Cell line Z could not be adapted to 0% serum > condition and hence we have unbalanced set of conditions. For clarity, > the conditions are listed below > > A - Y at 0% D - Z at 5% > B - Y at 5% E - Z at 10% > C - Y at 10% > > We have come up with 9 different pairwise comparisons based on the > scientific questions we want to answer. But performing so many pairwise > comparisons will lead to more liberal FDR values. Hence we want to > perform ANOVA analysis on all the five conditions. > > Based on section 3.2.6 which explains how to perform ANOVA-like > analysis. From what I understand, it contrasts each column with > intercept and will list any differences that are non-zero (B-A, C-A, > D-A, E-A). But ideally we want B-A, B-C, B-D, E-B, C-A, D-C, E-C, D-A, > or E-A comparisons in an ANOVA test. > > If anyone can suggest a method on edgeR to perform ANOVA tests, that > will be perfect. If not, can someone suggest other possible packages > that could help me do an ANOVA analysis on multiple groups. > > Regards > > > Yogender Kumar Gowtham > Graduate Research Assistant > Clemson University > Department of Bioengineering > 202 Biosystems Research Complex > Clemson, SC > (864)650-7451 ______________________________________________________________________ The information in this email is confidential and intend...{{dropped:4}}

Hello, It looks like you have already figured out how to perform an ANOVA- like test for differences among your five groups using edgeR. I believe your confusion is coming from the fact that the ANOVA-like test is only testing 4 contrasts, while you want to test 9 of them. The test only uses 4 contrasts because a test for differences among 5 groups has 4 degrees of freedom. The other five contrasts that you desire are actually just linear combinations of the first 4. Let's use the example you gave in your email, using group A as the intercept, so the 4 comparisons represented in the design are B-A, C-A, D-A, and E-A. If you wanted to calculate the C-B comparison, note that ((C-A) - (B-A)) = C - A - B + A = C - B. So, to get the logFC for C-B, you simply take the C-A logFC and subtract the B-A logFC. In a similar fashion, every other comparison can be computed in terms of the original 4 comparisons. Note that while only 4 comparisons are explicitly represented in the model, the ANOVA-like test is in fact testing for any evidence of differences between any of the 5 groups, which includes all 9 comparisons that you have specified, as well as the tenth comparison, E-D, which you didn't specify (there are 10 possible pairwise comparisons between 5 samples). -Ryan On 07/31/2014 03:52 PM, Yogender Gowtham wrote: > Hello All: > > I am Yogender Gowtham, a graduate student from Clemson University. We are > using RNA-Seq to investigate the transcriptional changes of Chinese Hamster > Ovary (CHO) cells during serum-free adaptation. Our experiment has two cell > lines, say Y and Z, under 3 different serum concentrations, say, 10%, 5% > and 0%. Cell line Z could not be adapted to 0% serum condition and hence we > have unbalanced set of conditions. For clarity, the conditions are listed > below > > A - Y at 0% D - Z at 5% > B - Y at 5% E - Z at 10% > C - Y at 10% > > We have come up with 9 different pairwise comparisons based on the > scientific questions we want to answer. But performing so many pairwise > comparisons will lead to more liberal FDR values. Hence we want to perform > ANOVA analysis on all the five conditions. > > Based on section 3.2.6 which explains how to perform ANOVA-like analysis. > >From what I understand, it contrasts each column with intercept and will > list any differences that are non-zero (B-A, C-A, D-A, E-A). But ideally we > want B-A, B-C, B-D, E-B, C-A, D-C, E-C, D-A, or E-A comparisons in an ANOVA > test. > > If anyone can suggest a method on edgeR to perform ANOVA tests, that will > be perfect. If not, can someone suggest other possible packages that could > help me do an ANOVA analysis on multiple groups. > > Regards > > > Yogender Kumar Gowtham > Graduate Research Assistant > Clemson University > Department of Bioengineering > 202 Biosystems Research Complex > Clemson, SC > (864)650-7451 > > [[alternative HTML version deleted]] > > _______________________________________________ > 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