RNA degradation plot with oligo package GeneFeatureSet objects
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heyi xiao ▴ 360
@heyi-xiao-3308
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Hi all, In affy package, I can use AffyRNAdeg and plotAffyRNAdeg to plot and check RNA degradation. Is there any way to do so in oligo package for GeneFeatureSet,which is equivalent to AffyBatch in affy package. I look at the GeneFeatureSet and AffyBatch, they quite similar. But not sure what can be done here. I can either modify AffyRNAdeg and plotAffyRNAdeg functions to fit them for GeneFeatureSet, or I can convert GeneFeatureSet to AffyBatch and use the affy package degradation functions. Any suggestions would be highly appreciated. Heyi
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@james-w-macdonald-5106
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Hi Heyi, On 8/14/2013 4:47 PM, heyi xiao wrote: > Hi all, > In affy package, I can use AffyRNAdeg and plotAffyRNAdeg to plot and check RNA degradation. Is there any way to do so in oligo package for GeneFeatureSet,which is equivalent to AffyBatch in affy package. I look at the GeneFeatureSet and AffyBatch, they quite similar. But not sure what can be done here. I can either modify AffyRNAdeg and plotAffyRNAdeg functions to fit them for GeneFeatureSet, or I can convert GeneFeatureSet to AffyBatch and use the affy package degradation functions. Any suggestions would be highly appreciated. While I suppose you could hypothetically do the conversion, I wonder if it makes conceptual sense. The 3'-biased Affy arrays were all based off an oligo-dT primer that was used to convert mRNA to cDNA, so the reverse transcription proceeded from the 3' end of the mRNA, always. In this case you can wonder about two things. First, how far did the RT step proceed? Did you in general get good RT all the way to the most 5' of the probes in the probesets? Second, since we were using the polyA tail at the 3' end, by definition the mRNA wasn't degraded from the 3' end. However, it might have had more or less extensive degradation from the 5' end, so the RT may have gone to completion, but the degradation had proceeded past the most 5' probes. So both things are confounded, as we cannot distinguish RT that didn't proceed too far from highly degraded mRNA, but no matter. What we could do is say how much signal we were getting from the more 5' probes, and decide if we wanted to do something about that (like only use the first 8 probes or whatever). For the newer generation of Affy arrays, we use a random primer, so the RT step proceeds from a random point in the transcript and proceeds towards the 5' end (at least I think it is still directional). Since the RT no longer starts from one end of the transcript, it is no longer clear what differential amounts of probe signal would actually signify. In addition, with the newer generation of Affy arrays, we can collapse the probes into different probesets, depending on what we are trying to measure (e.g., you can try to measure expression at the exon level or the transcript level). I think trying to do this would be more difficult than it would be worth, especially given that I don't know what you would do if you were to decide there had been degradation. Best, Jim > Heyi > > _______________________________________________ > 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 -- James W. MacDonald, M.S. Biostatistician University of Washington Environmental and Occupational Health Sciences 4225 Roosevelt Way NE, # 100 Seattle WA 98105-6099
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Hi Jim, Thanks for the informative notes. I really learned things about RNA degradation and affy array design! I see you what mean. But I only use RNA degradation as a quality assessment tool. I am less interested in estimate exactly how much RNA degradation happened in the RNA molecules in one sample/array, I am more interested in the different degradation patterns seen across different samples. Normally degradation curves for different samples stack together consistently and nicely. Even with the newer generation Affy arrays, an outlier degradation curve always suggest some quality issue, mostly likely RNA degradation. Such RNA degradation curves together with other quality check help me either kick out the problematic samples or have them redone. BTW, currently only oligo package seems to work with the new Ovine Gene 1.1 ST array, for which I don?t see an CDF package in bioconductor as other affy chip types. Therefore, I can?t go with affy and other packages which provide RNA degradation plots. Can I use makecdfenv package to build CDF package from PGF and CLF files? Heyi -------------------------------------------- On Fri, 8/16/13, James W. MacDonald <jmacdon at="" uw.edu=""> wrote: Subject: Re: [BioC] RNA degradation plot with oligo package GeneFeatureSet objects Cc: bioconductor at r-project.org Date: Friday, August 16, 2013, 12:52 PM Hi Heyi, On 8/14/2013 4:47 PM, heyi xiao wrote: > Hi all, > In affy package, I can use AffyRNAdeg and plotAffyRNAdeg to plot and check RNA degradation. Is there any way to do so in oligo package for GeneFeatureSet,which is equivalent to AffyBatch in affy package. I look at the GeneFeatureSet and AffyBatch, they quite similar. But not sure what can be done here. I can either modify AffyRNAdeg and plotAffyRNAdeg functions to fit them for GeneFeatureSet, or I can convert GeneFeatureSet to AffyBatch and use the affy package degradation functions. Any suggestions would be highly appreciated. While I suppose you could hypothetically do the conversion, I wonder if it makes conceptual sense. The 3'-biased Affy arrays were all based off an oligo-dT primer that was used to convert mRNA to cDNA, so the reverse transcription proceeded from the 3' end of the mRNA, always. In this case you can wonder about two things. First, how far did the RT step proceed? Did you in general get good RT all the way to the most 5' of the probes in the probesets? Second, since we were using the polyA tail at the 3' end, by definition the mRNA wasn't degraded from the 3' end. However, it might have had more or less extensive degradation from the 5' end, so the RT may have gone to completion, but the degradation had proceeded past the most 5' probes. So both things are confounded, as we cannot distinguish RT that didn't proceed too far from highly degraded mRNA, but no matter. What we could do is say how much signal we were getting from the more 5' probes, and decide if we wanted to do something about that (like only use the first 8 probes or whatever). For the newer generation of Affy arrays, we use a random primer, so the RT step proceeds from a random point in the transcript and proceeds towards the 5' end (at least I think it is still directional). Since the RT no longer starts from one end of the transcript, it is no longer clear what differential amounts of probe signal would actually signify. In addition, with the newer generation of Affy arrays, we can collapse the probes into different probesets, depending on what we are trying to measure (e.g., you can try to measure expression at the exon level or the transcript level). I think trying to do this would be more difficult than it would be worth, especially given that I don't know what you would do if you were to decide there had been degradation. Best, Jim > Heyi > > _______________________________________________ > 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 -- James W. MacDonald, M.S. Biostatistician University of Washington Environmental and Occupational Health Sciences 4225 Roosevelt Way NE, # 100 Seattle WA 98105-6099
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Hi Heyi, No you can't use the affy package for that chip type. As an aside, I have never found an array that I wanted to exclude based on the RNA degradation plot that I hadn't already decided to exclude based on either a PCA plot, a density plot of the raw data, or a NUSE or RLE plot. In other words, I think there are much better ways to find outlier plots than the degradation plot. Best, Jim On 8/16/2013 3:33 PM, heyi xiao wrote: > Hi Jim, > Thanks for the informative notes. I really learned things about RNA degradation and affy array design! > I see you what mean. But I only use RNA degradation as a quality assessment tool. I am less interested in estimate exactly how much RNA degradation happened in the RNA molecules in one sample/array, I am more interested in the different degradation patterns seen across different samples. Normally degradation curves for different samples stack together consistently and nicely. Even with the newer generation Affy arrays, an outlier degradation curve always suggest some quality issue, mostly likely RNA degradation. Such RNA degradation curves together with other quality check help me either kick out the problematic samples or have them redone. > BTW, currently only oligo package seems to work with the new Ovine Gene 1.1 ST array, for which I don?t see an CDF package in bioconductor as other affy chip types. Therefore, I can?t go with affy and other packages which provide RNA degradation plots. Can I use makecdfenv package to build CDF package from PGF and CLF files? > Heyi > > -------------------------------------------- > On Fri, 8/16/13, James W. MacDonald<jmacdon at="" uw.edu=""> wrote: > > Subject: Re: [BioC] RNA degradation plot with oligo package GeneFeatureSet objects > To: "heyi xiao"<xiaoheyiyh at="" yahoo.com=""> > Cc: bioconductor at r-project.org > Date: Friday, August 16, 2013, 12:52 PM > > Hi Heyi, > > On 8/14/2013 4:47 PM, heyi xiao wrote: > > Hi all, > > In affy package, I can use AffyRNAdeg and > plotAffyRNAdeg to plot and check RNA degradation. Is there > any way to do so in oligo package for GeneFeatureSet,which > is equivalent to AffyBatch in affy package. I look at the > GeneFeatureSet and AffyBatch, they quite similar. But not > sure what can be done here. I can either modify AffyRNAdeg > and plotAffyRNAdeg functions to fit them for GeneFeatureSet, > or I can convert GeneFeatureSet to AffyBatch and use the > affy package degradation functions. Any suggestions would be > highly appreciated. > > While I suppose you could hypothetically do the conversion, > I wonder if it makes conceptual sense. > > The 3'-biased Affy arrays were all based off an oligo-dT > primer that was used to convert mRNA to cDNA, so the reverse > transcription proceeded from the 3' end of the mRNA, always. > In this case you can wonder about two things. First, how far > did the RT step proceed? Did you in general get good RT all > the way to the most 5' of the probes in the probesets? > > Second, since we were using the polyA tail at the 3' end, by > definition the mRNA wasn't degraded from the 3' end. > However, it might have had more or less extensive > degradation from the 5' end, so the RT may have gone to > completion, but the degradation had proceeded past the most > 5' probes. > > So both things are confounded, as we cannot distinguish RT > that didn't proceed too far from highly degraded mRNA, but > no matter. What we could do is say how much signal we were > getting from the more 5' probes, and decide if we wanted to > do something about that (like only use the first 8 probes or > whatever). > > For the newer generation of Affy arrays, we use a random > primer, so the RT step proceeds from a random point in the > transcript and proceeds towards the 5' end (at least I think > it is still directional). Since the RT no longer starts from > one end of the transcript, it is no longer clear what > differential amounts of probe signal would actually > signify. > > In addition, with the newer generation of Affy arrays, we > can collapse the probes into different probesets, depending > on what we are trying to measure (e.g., you can try to > measure expression at the exon level or the transcript > level). > > I think trying to do this would be more difficult than it > would be worth, especially given that I don't know what you > would do if you were to decide there had been degradation. > > Best, > > Jim > > > > Heyi > > > > _______________________________________________ > > 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 > > -- James W. MacDonald, M.S. > Biostatistician > University of Washington > Environmental and Occupational Health Sciences > 4225 Roosevelt Way NE, # 100 > Seattle WA 98105-6099 > > -- James W. MacDonald, M.S. Biostatistician University of Washington Environmental and Occupational Health Sciences 4225 Roosevelt Way NE, # 100 Seattle WA 98105-6099
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Hi Jim, You mean, I can?t use makecdfenv package to build CDF package from PGF and CLF files? I will definitely check these other plots before decide on quality issue. Thanks! Heyi -------------------------------------------- On Fri, 8/16/13, James W. MacDonald <jmacdon at="" uw.edu=""> wrote: Subject: Re: [BioC] RNA degradation plot with oligo package GeneFeatureSet objects Cc: bioconductor at r-project.org Date: Friday, August 16, 2013, 3:59 PM Hi Heyi, No you can't use the affy package for that chip type. As an aside, I have never found an array that I wanted to exclude based on the RNA degradation plot that I hadn't already decided to exclude based on either a PCA plot, a density plot of the raw data, or a NUSE or RLE plot. In other words, I think there are much better ways to find outlier plots than the degradation plot. Best, Jim On 8/16/2013 3:33 PM, heyi xiao wrote: > Hi Jim, > Thanks for the informative notes. I really learned [[elided Yahoo spam]] > I see you what mean. But I only use RNA degradation as a quality assessment tool. I am less interested in estimate exactly how much RNA degradation happened in the RNA molecules in one sample/array, I am more interested in the different degradation patterns seen across different samples. Normally degradation curves for different samples stack together consistently and nicely. Even with the newer generation Affy arrays, an outlier degradation curve always suggest some quality issue, mostly likely RNA degradation. Such RNA degradation curves together with other quality check help me either kick out the problematic samples or have them redone. > BTW, currently only oligo package seems to work with the new Ovine Gene 1.1 ST array, for which I don?t see an CDF package in bioconductor as other affy chip types. Therefore, I can?t go with affy and other packages which provide RNA degradation plots. Can I use makecdfenv package to build CDF package from PGF and CLF files? > Heyi > > -------------------------------------------- > On Fri, 8/16/13, James W. MacDonald<jmacdon at="" uw.edu="">? wrote: > >???Subject: Re: [BioC] RNA degradation plot with oligo package GeneFeatureSet objects >???Cc: bioconductor at r-project.org >???Date: Friday, August 16, 2013, 12:52 PM > >???Hi Heyi, > >???On 8/14/2013 4:47 PM, heyi xiao wrote: >???>? Hi all, >???>? In affy package, I can use AffyRNAdeg and >???plotAffyRNAdeg to plot and check RNA degradation. Is there >???any way to do so in oligo package for GeneFeatureSet,which >???is equivalent to AffyBatch in affy package. I look at the >???GeneFeatureSet and AffyBatch, they quite similar. But not >???sure what can be done here. I can either modify AffyRNAdeg >???and plotAffyRNAdeg functions to fit them for GeneFeatureSet, >???or I can convert GeneFeatureSet to AffyBatch and use the >???affy package degradation functions. Any suggestions would be >???highly appreciated. > >???While I suppose you could hypothetically do the conversion, >???I wonder if it makes conceptual sense. > >???The 3'-biased Affy arrays were all based off an oligo-dT >???primer that was used to convert mRNA to cDNA, so the reverse >???transcription proceeded from the 3' end of the mRNA, always. >???In this case you can wonder about two things. First, how far >???did the RT step proceed? Did you in general get good RT all >???the way to the most 5' of the probes in the probesets? > >???Second, since we were using the polyA tail at the 3' end, by >???definition the mRNA wasn't degraded from the 3' end. >???However, it might have had more or less extensive >???degradation from the 5' end, so the RT may have gone to >???completion, but the degradation had proceeded past the most >???5' probes. > >???So both things are confounded, as we cannot distinguish RT >???that didn't proceed too far from highly degraded mRNA, but >???no matter. What we could do is say how much signal we were >???getting from the more 5' probes, and decide if we wanted to >???do something about that (like only use the first 8 probes or >???whatever). > >???For the newer generation of Affy arrays, we use a random >???primer, so the RT step proceeds from a random point in the >???transcript and proceeds towards the 5' end (at least I think >???it is still directional). Since the RT no longer starts from >???one end of the transcript, it is no longer clear what >???differential amounts of probe signal would actually >???signify. > >???In addition, with the newer generation of Affy arrays, we >???can collapse the probes into different probesets, depending >???on what we are trying to measure (e.g., you can try to >???measure expression at the exon level or the transcript >???level). > >???I think trying to do this would be more difficult than it >???would be worth, especially given that I don't know what you >???would do if you were to decide there had been degradation. > >???Best, > >???Jim > > >???>? Heyi >???> >???>? _______________________________________________ >???>? 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 > >???-- James W. MacDonald, M.S. >???Biostatistician >???University of Washington >???Environmental and Occupational Health Sciences >???4225 Roosevelt Way NE, # 100 >???Seattle WA 98105-6099 > > -- James W. MacDonald, M.S. Biostatistician University of Washington Environmental and Occupational Health Sciences 4225 Roosevelt Way NE, # 100 Seattle WA 98105-6099
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Hi Heyi, That's exactly what I mean. Best, Jim On 8/16/2013 6:26 PM, heyi xiao wrote: > Hi Jim, > You mean, I can?t use makecdfenv package to build CDF package from PGF and CLF files? > I will definitely check these other plots before decide on quality issue. > Thanks! > Heyi > > -------------------------------------------- > On Fri, 8/16/13, James W. MacDonald<jmacdon at="" uw.edu=""> wrote: > > Subject: Re: [BioC] RNA degradation plot with oligo package GeneFeatureSet objects > To: "heyi xiao"<xiaoheyiyh at="" yahoo.com=""> > Cc: bioconductor at r-project.org > Date: Friday, August 16, 2013, 3:59 PM > > Hi Heyi, > > No you can't use the affy package for that chip type. As an > aside, I > have never found an array that I wanted to exclude based on > the RNA > degradation plot that I hadn't already decided to exclude > based on > either a PCA plot, a density plot of the raw data, or a NUSE > or RLE > plot. In other words, I think there are much better ways to > find outlier > plots than the degradation plot. > > > Best, > > Jim > > > > On 8/16/2013 3:33 PM, heyi xiao wrote: > > Hi Jim, > > Thanks for the informative notes. I really learned > things about RNA degradation and affy array design! > > I see you what mean. But I only use RNA degradation as > a quality assessment tool. I am less interested in estimate > exactly how much RNA degradation happened in the RNA > molecules in one sample/array, I am more interested in the > different degradation patterns seen across different > samples. Normally degradation curves for different samples > stack together consistently and nicely. Even with the newer > generation Affy arrays, an outlier degradation curve always > suggest some quality issue, mostly likely RNA degradation. > Such RNA degradation curves together with other quality > check help me either kick out the problematic samples or > have them redone. > > BTW, currently only oligo package seems to work with > the new Ovine Gene 1.1 ST array, for which I don?t see an > CDF package in bioconductor as other affy chip types. > Therefore, I can?t go with affy and other packages which > provide RNA degradation plots. Can I use makecdfenv package > to build CDF package from PGF and CLF files? > > Heyi > > > > -------------------------------------------- > > On Fri, 8/16/13, James W. MacDonald<jmacdon at="" uw.edu=""> > wrote: > > > > Subject: Re: [BioC] RNA degradation > plot with oligo package GeneFeatureSet objects > > To: "heyi xiao"<xiaoheyiyh at="" yahoo.com=""> > > Cc: bioconductor at r-project.org > > Date: Friday, August 16, 2013, 12:52 > PM > > > > Hi Heyi, > > > > On 8/14/2013 4:47 PM, heyi xiao > wrote: > > > Hi all, > > > In affy package, I can use > AffyRNAdeg and > > plotAffyRNAdeg to plot and check RNA > degradation. Is there > > any way to do so in oligo package for > GeneFeatureSet,which > > is equivalent to AffyBatch in affy > package. I look at the > > GeneFeatureSet and AffyBatch, they > quite similar. But not > > sure what can be done here. I can > either modify AffyRNAdeg > > and plotAffyRNAdeg functions to fit > them for GeneFeatureSet, > > or I can convert GeneFeatureSet to > AffyBatch and use the > > affy package degradation functions. > Any suggestions would be > > highly appreciated. > > > > While I suppose you could > hypothetically do the conversion, > > I wonder if it makes conceptual > sense. > > > > The 3'-biased Affy arrays were all > based off an oligo-dT > > primer that was used to convert mRNA > to cDNA, so the reverse > > transcription proceeded from the 3' > end of the mRNA, always. > > In this case you can wonder about two > things. First, how far > > did the RT step proceed? Did you in > general get good RT all > > the way to the most 5' of the probes > in the probesets? > > > > Second, since we were using the polyA > tail at the 3' end, by > > definition the mRNA wasn't degraded > from the 3' end. > > However, it might have had more or > less extensive > > degradation from the 5' end, so the RT > may have gone to > > completion, but the degradation had > proceeded past the most > > 5' probes. > > > > So both things are confounded, as we > cannot distinguish RT > > that didn't proceed too far from > highly degraded mRNA, but > > no matter. What we could do is say how > much signal we were > > getting from the more 5' probes, and > decide if we wanted to > > do something about that (like only use > the first 8 probes or > > whatever). > > > > For the newer generation of Affy > arrays, we use a random > > primer, so the RT step proceeds from a > random point in the > > transcript and proceeds towards the 5' > end (at least I think > > it is still directional). Since the RT > no longer starts from > > one end of the transcript, it is no > longer clear what > > differential amounts of probe signal > would actually > > signify. > > > > In addition, with the newer generation > of Affy arrays, we > > can collapse the probes into different > probesets, depending > > on what we are trying to measure > (e.g., you can try to > > measure expression at the exon level > or the transcript > > level). > > > > I think trying to do this would be > more difficult than it > > would be worth, especially given that > I don't know what you > > would do if you were to decide there > had been degradation. > > > > Best, > > > > Jim > > > > > > > Heyi > > > > > > > _______________________________________________ > > > 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 > > > > -- James W. MacDonald, M.S. > > Biostatistician > > University of Washington > > Environmental and Occupational Health > Sciences > > 4225 Roosevelt Way NE, # 100 > > Seattle WA 98105-6099 > > > > > > -- > James W. MacDonald, M.S. > Biostatistician > University of Washington > Environmental and Occupational Health Sciences > 4225 Roosevelt Way NE, # 100 > Seattle WA 98105-6099 > > -- James W. MacDonald, M.S. Biostatistician University of Washington Environmental and Occupational Health Sciences 4225 Roosevelt Way NE, # 100 Seattle WA 98105-6099
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Dear Heyi, Function 'plotAffyRNAdeg()' of package 'xps' does allow you to plot RNA degradation plots for Whole Genome and Exon arrays, see Cahpter 5.4.1 of vignette 'xps.pdf'. I have done this for a couple of HuGene array data, and the results look interestingly. Especially there is a difference when you compare RNA degradation plots from frozen tissues vs paraffin embedded tissues. However, I am not sure how to interpret the results. Best regards, Christian _._._._._._._._._._._._._._._._._._ C.h.r.i.s.t.i.a.n S.t.r.a.t.o.w.a V.i.e.n.n.a A.u.s.t.r.i.a e.m.a.i.l: cstrato at aon.at _._._._._._._._._._._._._._._._._._ On 8/16/13 6:52 PM, James W. MacDonald wrote: > Hi Heyi, > > On 8/14/2013 4:47 PM, heyi xiao wrote: >> Hi all, >> In affy package, I can use AffyRNAdeg and plotAffyRNAdeg to plot and >> check RNA degradation. Is there any way to do so in oligo package for >> GeneFeatureSet,which is equivalent to AffyBatch in affy package. I >> look at the GeneFeatureSet and AffyBatch, they quite similar. But not >> sure what can be done here. I can either modify AffyRNAdeg and >> plotAffyRNAdeg functions to fit them for GeneFeatureSet, or I can >> convert GeneFeatureSet to AffyBatch and use the affy package >> degradation functions. Any suggestions would be highly appreciated. > > While I suppose you could hypothetically do the conversion, I wonder if > it makes conceptual sense. > > The 3'-biased Affy arrays were all based off an oligo-dT primer that was > used to convert mRNA to cDNA, so the reverse transcription proceeded > from the 3' end of the mRNA, always. In this case you can wonder about > two things. First, how far did the RT step proceed? Did you in general > get good RT all the way to the most 5' of the probes in the probesets? > > Second, since we were using the polyA tail at the 3' end, by definition > the mRNA wasn't degraded from the 3' end. However, it might have had > more or less extensive degradation from the 5' end, so the RT may have > gone to completion, but the degradation had proceeded past the most 5' > probes. > > So both things are confounded, as we cannot distinguish RT that didn't > proceed too far from highly degraded mRNA, but no matter. What we could > do is say how much signal we were getting from the more 5' probes, and > decide if we wanted to do something about that (like only use the first > 8 probes or whatever). > > For the newer generation of Affy arrays, we use a random primer, so the > RT step proceeds from a random point in the transcript and proceeds > towards the 5' end (at least I think it is still directional). Since the > RT no longer starts from one end of the transcript, it is no longer > clear what differential amounts of probe signal would actually signify. > > In addition, with the newer generation of Affy arrays, we can collapse > the probes into different probesets, depending on what we are trying to > measure (e.g., you can try to measure expression at the exon level or > the transcript level). > > I think trying to do this would be more difficult than it would be > worth, especially given that I don't know what you would do if you were > to decide there had been degradation. > > Best, > > Jim > > >> Heyi >> >> _______________________________________________ >> 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 >
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Hi Christian, Thanks for the suggestion and sharing of degradation plot experience. Does xps work with the Ovine Gene 1.1 ST array? I don?t have a CDF package. Heyi -------------------------------------------- On Fri, 8/16/13, cstrato <cstrato at="" aon.at=""> wrote: Subject: Re: [BioC] RNA degradation plot with oligo package GeneFeatureSet objects To: "James W. MacDonald" <jmacdon at="" uw.edu="">, "heyi xiao" <xiaoheyiyh at="" yahoo.com=""> Cc: bioconductor at r-project.org Date: Friday, August 16, 2013, 2:01 PM Dear Heyi, Function 'plotAffyRNAdeg()' of package 'xps' does allow you to plot RNA degradation plots for Whole Genome and Exon arrays, see Cahpter 5.4.1 of vignette 'xps.pdf'. I have done this for a couple of HuGene array data, and the results look interestingly. Especially there is a difference when you compare RNA degradation plots from frozen tissues vs paraffin embedded tissues. However, I am not sure how to interpret the results. Best regards, Christian _._._._._._._._._._._._._._._._._._ C.h.r.i.s.t.i.a.n???S.t.r.a.t.o.w.a V.i.e.n.n.a? ? ? ? ???A.u.s.t.r.i.a e.m.a.i.l:? ? ? ? cstrato at aon.at _._._._._._._._._._._._._._._._._._ On 8/16/13 6:52 PM, James W. MacDonald wrote: > Hi Heyi, > > On 8/14/2013 4:47 PM, heyi xiao wrote: >> Hi all, >> In affy package, I can use AffyRNAdeg and plotAffyRNAdeg to plot and >> check RNA degradation. Is there any way to do so in oligo package for >> GeneFeatureSet,which is equivalent to AffyBatch in affy package. I >> look at the GeneFeatureSet and AffyBatch, they quite similar. But not >> sure what can be done here. I can either modify AffyRNAdeg and >> plotAffyRNAdeg functions to fit them for GeneFeatureSet, or I can >> convert GeneFeatureSet to AffyBatch and use the affy package >> degradation functions. Any suggestions would be highly appreciated. > > While I suppose you could hypothetically do the conversion, I wonder if > it makes conceptual sense. > > The 3'-biased Affy arrays were all based off an oligo-dT primer that was > used to convert mRNA to cDNA, so the reverse transcription proceeded > from the 3' end of the mRNA, always. In this case you can wonder about > two things. First, how far did the RT step proceed? Did you in general > get good RT all the way to the most 5' of the probes in the probesets? > > Second, since we were using the polyA tail at the 3' end, by definition > the mRNA wasn't degraded from the 3' end. However, it might have had > more or less extensive degradation from the 5' end, so the RT may have > gone to completion, but the degradation had proceeded past the most 5' > probes. > > So both things are confounded, as we cannot distinguish RT that didn't > proceed too far from highly degraded mRNA, but no matter. What we could > do is say how much signal we were getting from the more 5' probes, and > decide if we wanted to do something about that (like only use the first > 8 probes or whatever). > > For the newer generation of Affy arrays, we use a random primer, so the > RT step proceeds from a random point in the transcript and proceeds > towards the 5' end (at least I think it is still directional). Since the > RT no longer starts from one end of the transcript, it is no longer > clear what differential amounts of probe signal would actually signify. > > In addition, with the newer generation of Affy arrays, we can collapse > the probes into different probesets, depending on what we are trying to > measure (e.g., you can try to measure expression at the exon level or > the transcript level). > > I think trying to do this would be more difficult than it would be > worth, especially given that I don't know what you would do if you were > to decide there had been degradation. > > Best, > > Jim > > >> Heyi >> >> _______________________________________________ >> 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 >
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Dear Heyi, Yes, xps should work with the Ovine Gene 1.1 ST arrays, since it does work with the Human, Mouse and Rat Gene 1.1 ST arrays. These arrays use the PGF-files from Affymetrix and 'xps/examples/script4schemes.R' shows you how to create the 'scheme' files for xps. Best regards, Christian On 8/16/13 9:36 PM, heyi xiao wrote: > Hi Christian, > Thanks for the suggestion and sharing of degradation plot experience. Does xps work with the Ovine Gene 1.1 ST array? I don?t have a CDF package. > Heyi > > -------------------------------------------- > On Fri, 8/16/13, cstrato <cstrato at="" aon.at=""> wrote: > > Subject: Re: [BioC] RNA degradation plot with oligo package GeneFeatureSet objects > To: "James W. MacDonald" <jmacdon at="" uw.edu="">, "heyi xiao" <xiaoheyiyh at="" yahoo.com=""> > Cc: bioconductor at r-project.org > Date: Friday, August 16, 2013, 2:01 PM > > Dear Heyi, > > Function 'plotAffyRNAdeg()' of package 'xps' does allow you > to plot RNA > degradation plots for Whole Genome and Exon arrays, see > Cahpter 5.4.1 of > vignette 'xps.pdf'. > > I have done this for a couple of HuGene array data, and the > results look > interestingly. Especially there is a difference when you > compare RNA > degradation plots from frozen tissues vs paraffin embedded > tissues. > However, I am not sure how to interpret the results. > > Best regards, > Christian > _._._._._._._._._._._._._._._._._._ > C.h.r.i.s.t.i.a.n S.t.r.a.t.o.w.a > V.i.e.n.n.a > A.u.s.t.r.i.a > e.m.a.i.l: cstrato at aon.at > _._._._._._._._._._._._._._._._._._ > > > > On 8/16/13 6:52 PM, James W. MacDonald wrote: > > Hi Heyi, > > > > On 8/14/2013 4:47 PM, heyi xiao wrote: > >> Hi all, > >> In affy package, I can use AffyRNAdeg and > plotAffyRNAdeg to plot and > >> check RNA degradation. Is there any way to do so in > oligo package for > >> GeneFeatureSet,which is equivalent to AffyBatch in > affy package. I > >> look at the GeneFeatureSet and AffyBatch, they > quite similar. But not > >> sure what can be done here. I can either modify > AffyRNAdeg and > >> plotAffyRNAdeg functions to fit them for > GeneFeatureSet, or I can > >> convert GeneFeatureSet to AffyBatch and use the > affy package > >> degradation functions. Any suggestions would be > highly appreciated. > > > > While I suppose you could hypothetically do the > conversion, I wonder if > > it makes conceptual sense. > > > > The 3'-biased Affy arrays were all based off an > oligo-dT primer that was > > used to convert mRNA to cDNA, so the reverse > transcription proceeded > > from the 3' end of the mRNA, always. In this case you > can wonder about > > two things. First, how far did the RT step proceed? Did > you in general > > get good RT all the way to the most 5' of the probes in > the probesets? > > > > Second, since we were using the polyA tail at the 3' > end, by definition > > the mRNA wasn't degraded from the 3' end. However, it > might have had > > more or less extensive degradation from the 5' end, so > the RT may have > > gone to completion, but the degradation had proceeded > past the most 5' > > probes. > > > > So both things are confounded, as we cannot distinguish > RT that didn't > > proceed too far from highly degraded mRNA, but no > matter. What we could > > do is say how much signal we were getting from the more > 5' probes, and > > decide if we wanted to do something about that (like > only use the first > > 8 probes or whatever). > > > > For the newer generation of Affy arrays, we use a > random primer, so the > > RT step proceeds from a random point in the transcript > and proceeds > > towards the 5' end (at least I think it is still > directional). Since the > > RT no longer starts from one end of the transcript, it > is no longer > > clear what differential amounts of probe signal would > actually signify. > > > > In addition, with the newer generation of Affy arrays, > we can collapse > > the probes into different probesets, depending on what > we are trying to > > measure (e.g., you can try to measure expression at the > exon level or > > the transcript level). > > > > I think trying to do this would be more difficult than > it would be > > worth, especially given that I don't know what you > would do if you were > > to decide there had been degradation. > > > > Best, > > > > Jim > > > > > >> Heyi > >> > >> _______________________________________________ > >> 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 > > > >
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Hi Christian, Great, I will check that out. Thanks for the info. Heyi -------------------------------------------- On Fri, 8/16/13, cstrato <cstrato at="" aon.at=""> wrote: Subject: Re: [BioC] RNA degradation plot with oligo package GeneFeatureSet objects Cc: bioconductor at r-project.org Date: Friday, August 16, 2013, 4:32 PM Dear Heyi, Yes, xps should work with the Ovine Gene 1.1 ST arrays, since it does work with the Human, Mouse and Rat Gene 1.1 ST arrays. These arrays use the PGF-files from Affymetrix and 'xps/examples/script4schemes.R' shows you how to create the 'scheme' files for xps. Best regards, Christian On 8/16/13 9:36 PM, heyi xiao wrote: > Hi Christian, > Thanks for the suggestion and sharing of degradation plot experience. Does xps work with the Ovine Gene 1.1 ST array? I don?t have a CDF package. > Heyi > > -------------------------------------------- > On Fri, 8/16/13, cstrato <cstrato at="" aon.at=""> wrote: > >???Subject: Re: [BioC] RNA degradation plot with oligo package GeneFeatureSet objects >???To: "James W. MacDonald" <jmacdon at="" uw.edu="">, >???Cc: bioconductor at r-project.org >???Date: Friday, August 16, 2013, 2:01 PM > >???Dear Heyi, > >???Function 'plotAffyRNAdeg()' of package 'xps' does allow you >???to plot RNA >???degradation plots for Whole Genome and Exon arrays, see >???Cahpter 5.4.1 of >???vignette 'xps.pdf'. > >???I have done this for a couple of HuGene array data, and the >???results look >???interestingly. Especially there is a difference when you >???compare RNA >???degradation plots from frozen tissues vs paraffin embedded >???tissues. >???However, I am not sure how to interpret the results. > >???Best regards, >???Christian >???_._._._._._._._._._._._._._._._._._ >???C.h.r.i.s.t.i.a.n???S.t.r.a.t.o.w.a >???V.i.e.n.n.a >? ? ? A.u.s.t.r.i.a >???e.m.a.i.l:? ? ? ? cstrato at aon.at >???_._._._._._._._._._._._._._._._._._ > > > >???On 8/16/13 6:52 PM, James W. MacDonald wrote: >???> Hi Heyi, >???> >???> On 8/14/2013 4:47 PM, heyi xiao wrote: >???>> Hi all, >???>> In affy package, I can use AffyRNAdeg and >???plotAffyRNAdeg to plot and >???>> check RNA degradation. Is there any way to do so in >???oligo package for >???>> GeneFeatureSet,which is equivalent to AffyBatch in >???affy package. I >???>> look at the GeneFeatureSet and AffyBatch, they >???quite similar. But not >???>> sure what can be done here. I can either modify >???AffyRNAdeg and >???>> plotAffyRNAdeg functions to fit them for >???GeneFeatureSet, or I can >???>> convert GeneFeatureSet to AffyBatch and use the >???affy package >???>> degradation functions. Any suggestions would be >???highly appreciated. >???> >???> While I suppose you could hypothetically do the >???conversion, I wonder if >???> it makes conceptual sense. >???> >???> The 3'-biased Affy arrays were all based off an >???oligo-dT primer that was >???> used to convert mRNA to cDNA, so the reverse >???transcription proceeded >???> from the 3' end of the mRNA, always. In this case you >???can wonder about >???> two things. First, how far did the RT step proceed? Did >???you in general >???> get good RT all the way to the most 5' of the probes in >???the probesets? >???> >???> Second, since we were using the polyA tail at the 3' >???end, by definition >???> the mRNA wasn't degraded from the 3' end. However, it >???might have had >???> more or less extensive degradation from the 5' end, so >???the RT may have >???> gone to completion, but the degradation had proceeded >???past the most 5' >???> probes. >???> >???> So both things are confounded, as we cannot distinguish >???RT that didn't >???> proceed too far from highly degraded mRNA, but no >???matter. What we could >???> do is say how much signal we were getting from the more >???5' probes, and >???> decide if we wanted to do something about that (like >???only use the first >???> 8 probes or whatever). >???> >???> For the newer generation of Affy arrays, we use a >???random primer, so the >???> RT step proceeds from a random point in the transcript >???and proceeds >???> towards the 5' end (at least I think it is still >???directional). Since the >???> RT no longer starts from one end of the transcript, it >???is no longer >???> clear what differential amounts of probe signal would >???actually signify. >???> >???> In addition, with the newer generation of Affy arrays, >???we can collapse >???> the probes into different probesets, depending on what >???we are trying to >???> measure (e.g., you can try to measure expression at the >???exon level or >???> the transcript level). >???> >???> I think trying to do this would be more difficult than >???it would be >???> worth, especially given that I don't know what you >???would do if you were >???> to decide there had been degradation. >???> >???> Best, >???> >???> Jim >???> >???> >???>> Heyi >???>> >???>> _______________________________________________ >???>> 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 >???> > >
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