Hello! I have been struggling with one of the skeletal muscle biopsies in my study. The RNA quality is very good and looking at tissue specific gene expression they are all there, although some with very different values for some genes compared the other biopsies. Please see the attached plots using edgeR. The gof() calculation gave 0 "TRUE" in the $outlier table. Can anyone shed some light on this? Thank you very much. -------------- next part -------------- A non-text attachment was scrubbed... Name: BCV-outlier.pdf Type: application/pdf Size: 119906 bytes Desc: not available URL: <https: stat.ethz.ch="" pipermail="" bioconductor="" attachments="" 20140302="" d360f67e="" attachment-0003.pdf=""> -------------- next part -------------- A non-text attachment was scrubbed... Name: MDS_plot-outlier.pdf Type: application/pdf Size: 4898 bytes Desc: not available URL: <https: stat.ethz.ch="" pipermail="" bioconductor="" attachments="" 20140302="" d360f67e="" attachment-0004.pdf=""> -------------- next part -------------- A non-text attachment was scrubbed... Name: GOF_plot-outlier.pdf Type: application/pdf Size: 1214490 bytes Desc: not available URL: <https: stat.ethz.ch="" pipermail="" bioconductor="" attachments="" 20140302="" d360f67e="" attachment-0005.pdf="">

That BCV plot looks bizarre, with the two bands. What does the BCV plot look like if you exclude the outlier sample? Does the double-banding go away? Does the prior.df change? Regardless, I think it's pretty clear from the MDS that this sample is an outlier and should probably be excluded from your analysis. On Sun Mar 2 12:00:50 2014, Sindre Lee wrote: > Hello! > I have been struggling with one of the skeletal muscle biopsies in my > study. The RNA quality is very good and looking at tissue specific > gene expression they are all there, although some with very different > values for some genes compared the other biopsies. > > Please see the attached plots using edgeR. The gof() calculation gave > 0 "TRUE" in the $outlier table. > > > Can anyone shed some light on this? Thank you very much. > > > _______________________________________________ > 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

Ok, since the upper band disappears when you exclude the outlier sample, I think that means that all the genes in the upper band of the original BCV plot are the specific genes that are responsible for the sample being an outlier, and the rest of the genes (the lower band) are behaving normally even in the outlier sample. You might try looking at the behavior of the 100 highest-dispersion genes in the outlier sample vs the other samples, to see if there is a consistent pattern (e.g. they are all way overrepresented in the outlier sample). Again, though, I'm not sure what you can do to fix the problem other than discarding the sample entirely. On Mon Mar 3 00:11:26 2014, Sindre Lee wrote: > On 2014-03-03 09:01, Ryan wrote: >> That BCV plot looks bizarre, with the two bands. What does the BCV >> plot look like if you exclude the outlier sample? Does the >> double-banding go away? Does the prior.df change? >> >> Regardless, I think it's pretty clear from the MDS that this sample >> is an outlier and should probably be excluded from your analysis. >> >> On Sun Mar 2 12:00:50 2014, Sindre Lee wrote: >>> Hello! >>> I have been struggling with one of the skeletal muscle biopsies in my >>> study. The RNA quality is very good and looking at tissue specific >>> gene expression they are all there, although some with very different >>> values for some genes compared the other biopsies. >>> >>> Please see the attached plots using edgeR. The gof() calculation gave >>> 0 "TRUE" in the $outlier table. >>> >>> >>> Can anyone shed some light on this? Thank you very much. >>> >>> >>> _______________________________________________ >>> 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 > > Thank you! > > Yes, it goes away after removing the outlier (see attachment). >

Hi Sindre, It seems that you have one suspicious sample in your data according to your MDS plot. That sample (on the very right of the MDS plot) is very likely to be an outlier, hence vastly increases your dispersion estimates. For statistical analysis purpose, it would be better to take that sample out. Regards, Yunshun Chen Message: 4 Date: Sun, 02 Mar 2014 21:00:50 +0100 From: Sindre Lee <sindre.lee@studmed.uio.no> To: <bioconductor at="" r-project.org=""> Subject: [BioC] edgeR: plotBCV, gof() and plotMDS, for outlier detection Message-ID: <b618869c628acccf65b86037c4774909 at="" ulrik.uio.no=""> Content-Type: text/plain; charset="utf-8"; Format="flowed" Hello! I have been struggling with one of the skeletal muscle biopsies in my study. The RNA quality is very good and looking at tissue specific gene expression they are all there, although some with very different values for some genes compared the other biopsies. Please see the attached plots using edgeR. The gof() calculation gave 0 "TRUE" in the $outlier table. Can anyone shed some light on this? Thank you very much. -------------- next part -------------- A non-text attachment was scrubbed... Name: BCV-outlier.pdf Type: application/pdf Size: 119906 bytes Desc: not available URL: <https: stat.ethz.ch="" pipermail="" bioconductor="" attachments="" 20140302="" d360="" f67e="" a="" ttachment.pdf=""> -------------- next part -------------- A non-text attachment was scrubbed... Name: MDS_plot-outlier.pdf Type: application/pdf Size: 4898 bytes Desc: not available URL: <https: stat.ethz.ch="" pipermail="" bioconductor="" attachments="" 20140302="" d360="" f67e="" a="" ttachment-0001.pdf=""> -------------- next part -------------- A non-text attachment was scrubbed... Name: GOF_plot-outlier.pdf Type: application/pdf Size: 1214490 bytes Desc: not available URL: <https: stat.ethz.ch="" pipermail="" bioconductor="" attachments="" 20140302="" d360="" f67e="" a="" ttachment-0002.pdf=""> ------------------------------ _______________________________________________ Bioconductor mailing list Bioconductor at r-project.org https://stat.ethz.ch/mailman/listinfo/bioconductor End of Bioconductor Digest, Vol 133, Issue 3 ******************************************** ______________________________________________________________________ The information in this email is confidential and intend...{{dropped:4}}