DESeq2 transcript level
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@alicia-r-perez-porro-5953
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Dear Bioconductor users, I am working with differential expression at the transcript (isoform) level. I have 6 different conditions (2 replicates/condition). I want to know if I can use DESeq2 for that or if the package can only be used at the gene level. Thanks, Alicia -- Alicia R. Pérez-Porro PhD candidate Giribet lab Department of Organismic and Evolutionary Biology MCZ labs Harvard University 26 Oxford St, Cambridge MA 02138 phone: +1 617-496-5308 fax: +1 617-495-5667 www.oeb.harvard.edu/faculty/giribet/ Department of Marine Ecology Center for Advanced Studies of Blanes (CEAB-CSIC) C/Accés Cala St. Francesc 14 17300 Blanes, Girona, SPAIN phone: +34 972 336 101 fax: +34 972 337 806 www.ceab.csic.es [[alternative HTML version deleted]]
DESeq2 DESeq2 • 5.8k views
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@mikelove
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hi Alicia, DESeq2 is intended for gene-level analysis. here is some explanation from Simon on the list as to why doing a count-based analysis at the transcript/isoform level is problematic: https://stat.ethz.ch/pipermail/bioconductor/2012-February/043410.html if you are interested in looking for differential exon usage, you can instead consider using the DEXSeq package: publication http://www.ncbi.nlm.nih.gov/pubmed/22722343 software: http://bioconductor.org/packages/release/bioc/html/DEXSeq.html Mike On Thu, May 8, 2014 at 7:26 PM, Alicia R. Pérez-Porro < alicia.r.perezporro@gmail.com> wrote: > Dear Bioconductor users, > > I am working with differential expression at the transcript (isoform) > level. I have 6 different conditions (2 replicates/condition). > > I want to know if I can use DESeq2 for that or if the package can only be > used at the gene level. > > Thanks, > Alicia > > > -- > Alicia R. Pérez-Porro > PhD candidate > > Giribet lab > Department of Organismic and Evolutionary Biology > MCZ labs > Harvard University > 26 Oxford St, Cambridge MA 02138 > phone: +1 617-496-5308 > fax: +1 617-495-5667 > www.oeb.harvard.edu/faculty/giribet/ > > Department of Marine Ecology > Center for Advanced Studies of Blanes (CEAB-CSIC) > C/Accés Cala St. Francesc 14 > 17300 Blanes, Girona, SPAIN > phone: +34 972 336 101 > fax: +34 972 337 806 > www.ceab.csic.es > > [[alternative HTML version deleted]] > > > _______________________________________________ > 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]]
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Hi Mike, Thanks for your answer. I read the explanation from Simon and am still a little bit confuse, reading the edgeR manual I found this: "*edgeR can be applied to di fferential expression at the gene, exon, transcript or tag level. In fact, read counts can be summarized by any genomic feature. edgeR analyses at the exon level are easily extended to detect di fferential splicing or isoform-specifi c dif ferential **expression*". So, my initial idea was to treat each isoform like a gene because for the approach that I am attempting right now I am not interested in differences in expression of isoforms belonging to the same gene. I did my *de novo*with Trinity, alignment using Bowtie and estimation of abundance with RSEM. I created a matrix with the RSEM counts (isoforms results) and I was planning on using it as input for my next step. I understand that even I want to treat each isoform as a gene to do it with DESeq or DESeq2 is not a good idea. I really don't know about edgeR and am also considering EBseq. Suggestions? Thoughts? Thank you in advance. Alicia -- Alicia R. Pérez-Porro PhD candidate Giribet lab Department of Organismic and Evolutionary Biology MCZ labs Harvard University 26 Oxford St, Cambridge MA 02138 phone: +1 617-496-5308 fax: +1 617-495-5667 www.oeb.harvard.edu/faculty/giribet/ Department of Marine Ecology Center for Advanced Studies of Blanes (CEAB-CSIC) C/Accés Cala St. Francesc 14 17300 Blanes, Girona, SPAIN phone: +34 972 336 101 fax: +34 972 337 806 www.ceab.csic.es On Fri, May 9, 2014 at 8:07 AM, Michael Love <michaelisaiahlove@gmail.com>wrote: > hi Alicia, > > DESeq2 is intended for gene-level analysis. here is some explanation from > Simon on the list as to why doing a count-based analysis at the > transcript/isoform level is problematic: > > https://stat.ethz.ch/pipermail/bioconductor/2012-February/043410.html > > if you are interested in looking for differential exon usage, you can > instead consider using the DEXSeq package: > > publication > http://www.ncbi.nlm.nih.gov/pubmed/22722343 > > software: > http://bioconductor.org/packages/release/bioc/html/DEXSeq.html > > Mike > > > > On Thu, May 8, 2014 at 7:26 PM, Alicia R. Pérez-Porro < > alicia.r.perezporro@gmail.com> wrote: > >> Dear Bioconductor users, >> >> I am working with differential expression at the transcript (isoform) >> level. I have 6 different conditions (2 replicates/condition). >> >> I want to know if I can use DESeq2 for that or if the package can only be >> used at the gene level. >> >> Thanks, >> Alicia >> >> >> -- >> Alicia R. Pérez-Porro >> PhD candidate >> >> Giribet lab >> Department of Organismic and Evolutionary Biology >> MCZ labs >> Harvard University >> 26 Oxford St, Cambridge MA 02138 >> phone: +1 617-496-5308 >> fax: +1 617-495-5667 >> www.oeb.harvard.edu/faculty/giribet/ >> >> Department of Marine Ecology >> Center for Advanced Studies of Blanes (CEAB-CSIC) >> C/Accés Cala St. Francesc 14 >> 17300 Blanes, Girona, SPAIN >> phone: +34 972 336 101 >> fax: +34 972 337 806 >> www.ceab.csic.es >> >> [[alternative HTML version deleted]] >> >> >> _______________________________________________ >> 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]]
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hi Alicia, In your previous email, you didn't mention that you had used software for estimating isoform specific counts, so I pasted a link to Simon's answer concerning isoform counts. If one can assign reads to isoforms with high confidence then it would be fine to do testing with DESeq2. However, this is typically a difficult task which involves uncertainty in estimation and DESeq2 does not take into account any uncertainty in these estimated counts. If you have 10 reads in the count matrix, DESeq2 will take this to mean 10 reads were unambiguously assigned to this feature. Through estimation steps, this could be 10 reads plus or minus 10 reads. So the quality of the results will depend on the quality of the input. Mike On Fri, May 9, 2014 at 11:11 AM, Alicia R. P?rez-Porro <alicia.r.perezporro at="" gmail.com=""> wrote: > Hi Mike, > > Thanks for your answer. I read the explanation from Simon and am still a > little bit confuse, reading the edgeR manual I found this: "edgeR can be > applied to di fferential expression at the gene, exon, transcript or tag > level. In fact, read counts can be summarized by any genomic feature. edgeR > analyses at the exon level are easily extended to detect di fferential > splicing or isoform-specifi c dif ferential expression". > > So, my initial idea was to treat each isoform like a gene because for the > approach that I am attempting right now I am not interested in differences > in expression of isoforms belonging to the same gene. I did my de novo with > Trinity, alignment using Bowtie and estimation of abundance with RSEM. I > created a matrix with the RSEM counts (isoforms results) and I was planning > on using it as input for my next step. > > I understand that even I want to treat each isoform as a gene to do it with > DESeq or DESeq2 is not a good idea. I really don't know about edgeR and am > also considering EBseq. > > Suggestions? Thoughts? > > Thank you in advance. > Alicia > > > > -- > Alicia R. P?rez-Porro > PhD candidate > > Giribet lab > Department of Organismic and Evolutionary Biology > MCZ labs > Harvard University > 26 Oxford St, Cambridge MA 02138 > phone: +1 617-496-5308 > fax: +1 617-495-5667 > www.oeb.harvard.edu/faculty/giribet/ > > Department of Marine Ecology > Center for Advanced Studies of Blanes (CEAB-CSIC) > C/Acc?s Cala St. Francesc 14 > 17300 Blanes, Girona, SPAIN > phone: +34 972 336 101 > fax: +34 972 337 806 > www.ceab.csic.es > > > On Fri, May 9, 2014 at 8:07 AM, Michael Love <michaelisaiahlove at="" gmail.com=""> > wrote: >> >> hi Alicia, >> >> DESeq2 is intended for gene-level analysis. here is some explanation from >> Simon on the list as to why doing a count-based analysis at the >> transcript/isoform level is problematic: >> >> https://stat.ethz.ch/pipermail/bioconductor/2012-February/043410.html >> >> if you are interested in looking for differential exon usage, you can >> instead consider using the DEXSeq package: >> >> publication >> http://www.ncbi.nlm.nih.gov/pubmed/22722343 >> >> software: >> http://bioconductor.org/packages/release/bioc/html/DEXSeq.html >> >> Mike >> >> >> >> On Thu, May 8, 2014 at 7:26 PM, Alicia R. P?rez-Porro >> <alicia.r.perezporro at="" gmail.com=""> wrote: >>> >>> Dear Bioconductor users, >>> >>> I am working with differential expression at the transcript (isoform) >>> level. I have 6 different conditions (2 replicates/condition). >>> >>> I want to know if I can use DESeq2 for that or if the package can only be >>> used at the gene level. >>> >>> Thanks, >>> Alicia >>> >>> >>> -- >>> Alicia R. P?rez-Porro >>> PhD candidate >>> >>> Giribet lab >>> Department of Organismic and Evolutionary Biology >>> MCZ labs >>> Harvard University >>> 26 Oxford St, Cambridge MA 02138 >>> phone: +1 617-496-5308 >>> fax: +1 617-495-5667 >>> www.oeb.harvard.edu/faculty/giribet/ >>> >>> Department of Marine Ecology >>> Center for Advanced Studies of Blanes (CEAB-CSIC) >>> C/Acc?s Cala St. Francesc 14 >>> 17300 Blanes, Girona, SPAIN >>> phone: +34 972 336 101 >>> fax: +34 972 337 806 >>> www.ceab.csic.es >>> >>> [[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 >> >> >
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Hi everyone, Yes, sorry Mike, I forgot to mention that I calculated my isoform counts estimation with RSEM. Thomas, I am aware about the pipeline using RSEM+(edgeR or DESeq) to do DE analysis inside Trinity. I tried and I didn't like just because is not a good option to use with my data. I have non standard data and the pipeline is too hands off so works nicely if you have more standard data. Mainly what I want is to do pair-wise comparisons between my 5 different conditions (5 different moments along the life cycle of my animal) to generate a list of the most DEG, blast them to see what they are, potentially get the GO:terms associated to them and have an overview of what is happening along the life cycle. My intention in the future is probably continue with analysis in differential exon expression (DEXseq) and/or differential isoform expression. And I am also considering time-scale differential expression analysis. But like I said I'm planning on doing that in the future because right now I am interested just in an overview of what is going on in the life cycle of my animal. That's why I was thinking about treating the isoforms as genes. Does that make sense to you, guys? Thanks again, Alicia -- Alicia R. Pérez-Porro PhD candidate Giribet lab Department of Organismic and Evolutionary Biology MCZ labs Harvard University 26 Oxford St, Cambridge MA 02138 phone: +1 617-496-5308 fax: +1 617-495-5667 www.oeb.harvard.edu/faculty/giribet/ Department of Marine Ecology Center for Advanced Studies of Blanes (CEAB-CSIC) C/Accés Cala St. Francesc 14 17300 Blanes, Girona, SPAIN phone: +34 972 336 101 fax: +34 972 337 806 www.ceab.csic.es On Fri, May 9, 2014 at 1:45 PM, Michael Love <michaelisaiahlove@gmail.com>wrote: > hi Alicia, > > In your previous email, you didn't mention that you had used software > for estimating isoform specific counts, so I pasted a link to Simon's > answer concerning isoform counts. If one can assign reads to isoforms > with high confidence then it would be fine to do testing with DESeq2. > However, this is typically a difficult task which involves uncertainty > in estimation and DESeq2 does not take into account any uncertainty in > these estimated counts. If you have 10 reads in the count matrix, > DESeq2 will take this to mean 10 reads were unambiguously assigned to > this feature. Through estimation steps, this could be 10 reads plus or > minus 10 reads. So the quality of the results will depend on the > quality of the input. > > Mike > > On Fri, May 9, 2014 at 11:11 AM, Alicia R. Pérez-Porro > <alicia.r.perezporro@gmail.com> wrote: > > Hi Mike, > > > > Thanks for your answer. I read the explanation from Simon and am still a > > little bit confuse, reading the edgeR manual I found this: "edgeR can be > > applied to di fferential expression at the gene, exon, transcript or tag > > level. In fact, read counts can be summarized by any genomic feature. > edgeR > > analyses at the exon level are easily extended to detect di fferential > > splicing or isoform-specifi c dif ferential expression". > > > > So, my initial idea was to treat each isoform like a gene because for the > > approach that I am attempting right now I am not interested in > differences > > in expression of isoforms belonging to the same gene. I did my de novo > with > > Trinity, alignment using Bowtie and estimation of abundance with RSEM. I > > created a matrix with the RSEM counts (isoforms results) and I was > planning > > on using it as input for my next step. > > > > I understand that even I want to treat each isoform as a gene to do it > with > > DESeq or DESeq2 is not a good idea. I really don't know about edgeR and > am > > also considering EBseq. > > > > Suggestions? Thoughts? > > > > Thank you in advance. > > Alicia > > > > > > > > -- > > Alicia R. Pérez-Porro > > PhD candidate > > > > Giribet lab > > Department of Organismic and Evolutionary Biology > > MCZ labs > > Harvard University > > 26 Oxford St, Cambridge MA 02138 > > phone: +1 617-496-5308 > > fax: +1 617-495-5667 > > www.oeb.harvard.edu/faculty/giribet/ > > > > Department of Marine Ecology > > Center for Advanced Studies of Blanes (CEAB-CSIC) > > C/Accés Cala St. Francesc 14 > > 17300 Blanes, Girona, SPAIN > > phone: +34 972 336 101 > > fax: +34 972 337 806 > > www.ceab.csic.es > > > > > > On Fri, May 9, 2014 at 8:07 AM, Michael Love < > michaelisaiahlove@gmail.com> > > wrote: > >> > >> hi Alicia, > >> > >> DESeq2 is intended for gene-level analysis. here is some explanation > from > >> Simon on the list as to why doing a count-based analysis at the > >> transcript/isoform level is problematic: > >> > >> https://stat.ethz.ch/pipermail/bioconductor/2012-February/043410.html > >> > >> if you are interested in looking for differential exon usage, you can > >> instead consider using the DEXSeq package: > >> > >> publication > >> http://www.ncbi.nlm.nih.gov/pubmed/22722343 > >> > >> software: > >> http://bioconductor.org/packages/release/bioc/html/DEXSeq.html > >> > >> Mike > >> > >> > >> > >> On Thu, May 8, 2014 at 7:26 PM, Alicia R. Pérez-Porro > >> <alicia.r.perezporro@gmail.com> wrote: > >>> > >>> Dear Bioconductor users, > >>> > >>> I am working with differential expression at the transcript (isoform) > >>> level. I have 6 different conditions (2 replicates/condition). > >>> > >>> I want to know if I can use DESeq2 for that or if the package can only > be > >>> used at the gene level. > >>> > >>> Thanks, > >>> Alicia > >>> > >>> > >>> -- > >>> Alicia R. Pérez-Porro > >>> PhD candidate > >>> > >>> Giribet lab > >>> Department of Organismic and Evolutionary Biology > >>> MCZ labs > >>> Harvard University > >>> 26 Oxford St, Cambridge MA 02138 > >>> phone: +1 617-496-5308 > >>> fax: +1 617-495-5667 > >>> www.oeb.harvard.edu/faculty/giribet/ > >>> > >>> Department of Marine Ecology > >>> Center for Advanced Studies of Blanes (CEAB-CSIC) > >>> C/Accés Cala St. Francesc 14 > >>> 17300 Blanes, Girona, SPAIN > >>> phone: +34 972 336 101 > >>> fax: +34 972 337 806 > >>> www.ceab.csic.es > >>> > >>> [[alternative HTML version deleted]] > >>> > >>> > >>> _______________________________________________ > >>> 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]]
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Hi Alicia, Here is my two cents. I think RSEM isoform counts contain ?multi- isoform? reads, i.e. reads mapped to multiple isoforms, but from a single gene, so I don?t think DESeq2 would demonstrate it?s power in terms of accuracy in your case. If your purpose is only to obtain an ?inspiration of biology? from your data, why not simply use the fold change of your isoform expression and to check out what?s the most changed ones? Yes, you probably need some kind of normalization to leverage the library sizes. Cheers, Yuan On May 9, 2014, at 2:08 PM, Alicia R. P?rez-Porro <alicia.r.perezporro at="" gmail.com=""> wrote: > Hi everyone, > > Yes, sorry Mike, I forgot to mention that I calculated my isoform counts > estimation with RSEM. > > Thomas, I am aware about the pipeline using RSEM+(edgeR or DESeq) to do DE > analysis inside Trinity. I tried and I didn't like just because is not a > good option to use with my data. I have non standard data and the pipeline > is too hands off so works nicely if you have more standard data. > > Mainly what I want is to do pair-wise comparisons between my 5 different > conditions (5 different moments along the life cycle of my animal) to > generate a list of the most DEG, blast them to see what they are, > potentially get the GO:terms associated to them and have an overview of > what is happening along the life cycle. My intention in the future is > probably continue with analysis in differential exon expression (DEXseq) > and/or differential isoform expression. And I am also considering > time-scale differential expression analysis. But like I said I'm planning > on doing that in the future because right now I am interested just in an > overview of what is going on in the life cycle of my animal. That's why I > was thinking about treating the isoforms as genes. > > Does that make sense to you, guys? > > Thanks again, > Alicia > > > > -- > Alicia R. P?rez-Porro > PhD candidate > > Giribet lab > Department of Organismic and Evolutionary Biology > MCZ labs > Harvard University > 26 Oxford St, Cambridge MA 02138 > phone: +1 617-496-5308 > fax: +1 617-495-5667 > www.oeb.harvard.edu/faculty/giribet/ > > Department of Marine Ecology > Center for Advanced Studies of Blanes (CEAB-CSIC) > C/Acc?s Cala St. Francesc 14 > 17300 Blanes, Girona, SPAIN > phone: +34 972 336 101 > fax: +34 972 337 806 > www.ceab.csic.es > > > On Fri, May 9, 2014 at 1:45 PM, Michael Love <michaelisaiahlove at="" gmail.com="">wrote: > >> hi Alicia, >> >> In your previous email, you didn't mention that you had used software >> for estimating isoform specific counts, so I pasted a link to Simon's >> answer concerning isoform counts. If one can assign reads to isoforms >> with high confidence then it would be fine to do testing with DESeq2. >> However, this is typically a difficult task which involves uncertainty >> in estimation and DESeq2 does not take into account any uncertainty in >> these estimated counts. If you have 10 reads in the count matrix, >> DESeq2 will take this to mean 10 reads were unambiguously assigned to >> this feature. Through estimation steps, this could be 10 reads plus or >> minus 10 reads. So the quality of the results will depend on the >> quality of the input. >> >> Mike >> >> On Fri, May 9, 2014 at 11:11 AM, Alicia R. P?rez-Porro >> <alicia.r.perezporro at="" gmail.com=""> wrote: >>> Hi Mike, >>> >>> Thanks for your answer. I read the explanation from Simon and am still a >>> little bit confuse, reading the edgeR manual I found this: "edgeR can be >>> applied to di fferential expression at the gene, exon, transcript or tag >>> level. In fact, read counts can be summarized by any genomic feature. >> edgeR >>> analyses at the exon level are easily extended to detect di fferential >>> splicing or isoform-specifi c dif ferential expression". >>> >>> So, my initial idea was to treat each isoform like a gene because for the >>> approach that I am attempting right now I am not interested in >> differences >>> in expression of isoforms belonging to the same gene. I did my de novo >> with >>> Trinity, alignment using Bowtie and estimation of abundance with RSEM. I >>> created a matrix with the RSEM counts (isoforms results) and I was >> planning >>> on using it as input for my next step. >>> >>> I understand that even I want to treat each isoform as a gene to do it >> with >>> DESeq or DESeq2 is not a good idea. I really don't know about edgeR and >> am >>> also considering EBseq. >>> >>> Suggestions? Thoughts? >>> >>> Thank you in advance. >>> Alicia >>> >>> >>> >>> -- >>> Alicia R. P?rez-Porro >>> PhD candidate >>> >>> Giribet lab >>> Department of Organismic and Evolutionary Biology >>> MCZ labs >>> Harvard University >>> 26 Oxford St, Cambridge MA 02138 >>> phone: +1 617-496-5308 >>> fax: +1 617-495-5667 >>> www.oeb.harvard.edu/faculty/giribet/ >>> >>> Department of Marine Ecology >>> Center for Advanced Studies of Blanes (CEAB-CSIC) >>> C/Acc?s Cala St. Francesc 14 >>> 17300 Blanes, Girona, SPAIN >>> phone: +34 972 336 101 >>> fax: +34 972 337 806 >>> www.ceab.csic.es >>> >>> >>> On Fri, May 9, 2014 at 8:07 AM, Michael Love < >> michaelisaiahlove at gmail.com> >>> wrote: >>>> >>>> hi Alicia, >>>> >>>> DESeq2 is intended for gene-level analysis. here is some explanation >> from >>>> Simon on the list as to why doing a count-based analysis at the >>>> transcript/isoform level is problematic: >>>> >>>> https://stat.ethz.ch/pipermail/bioconductor/2012-February/043410.html >>>> >>>> if you are interested in looking for differential exon usage, you can >>>> instead consider using the DEXSeq package: >>>> >>>> publication >>>> http://www.ncbi.nlm.nih.gov/pubmed/22722343 >>>> >>>> software: >>>> http://bioconductor.org/packages/release/bioc/html/DEXSeq.html >>>> >>>> Mike >>>> >>>> >>>> >>>> On Thu, May 8, 2014 at 7:26 PM, Alicia R. P?rez-Porro >>>> <alicia.r.perezporro at="" gmail.com=""> wrote: >>>>> >>>>> Dear Bioconductor users, >>>>> >>>>> I am working with differential expression at the transcript (isoform) >>>>> level. I have 6 different conditions (2 replicates/condition). >>>>> >>>>> I want to know if I can use DESeq2 for that or if the package can only >> be >>>>> used at the gene level. >>>>> >>>>> Thanks, >>>>> Alicia >>>>> >>>>> >>>>> -- >>>>> Alicia R. P?rez-Porro >>>>> PhD candidate >>>>> >>>>> Giribet lab >>>>> Department of Organismic and Evolutionary Biology >>>>> MCZ labs >>>>> Harvard University >>>>> 26 Oxford St, Cambridge MA 02138 >>>>> phone: +1 617-496-5308 >>>>> fax: +1 617-495-5667 >>>>> www.oeb.harvard.edu/faculty/giribet/ >>>>> >>>>> Department of Marine Ecology >>>>> Center for Advanced Studies of Blanes (CEAB-CSIC) >>>>> C/Acc?s Cala St. Francesc 14 >>>>> 17300 Blanes, Girona, SPAIN >>>>> phone: +34 972 336 101 >>>>> fax: +34 972 337 806 >>>>> www.ceab.csic.es >>>>> >>>>> [[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 >>>> >>>> >>> >> > > [[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
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Hi Yuan, I emailed the RSEM group to make sure that RSEM isoform counts contain 'multi-isoform' reads. Here is the answer: *"Yes, RSEM explicitly handles reads that map to multiple isoforms in the same way that handles reads that map to multiple genes. eXpress essentially uses the same model as RSEM but uses different computational techniques to arrive at the same sort of quantification results, so I don't see it as being better than RSEM in this respect.* *The trouble stems from the fact that DESeq assumes that the counts assigned to each gene or transcript are observed, whereas multi- isoform or multi-gene reads violate this assumption, and thus the counts reported by RSEM or eXpress are only *estimates* of the counts for each gene/transcript and there is, of course, uncertainty associated with these estimates. DESeq does not explicitly take into account such uncertainty.* *Transcript-level differential expression methodology is tricky and is an active area of research. If you would like to continue with using RSEM for the quantification step, you might explore using EBSeq (bundled with RSEM and also available via bioconductor) for your differential expression analyses because it was specifically designed to accommodate increased uncertainty in counts for transcripts from genes with larger numbers of possible isoforms.* *Best,* *Colin"* -- Alicia R. Pérez-Porro PhD candidate Giribet lab Department of Organismic and Evolutionary Biology MCZ labs Harvard University 26 Oxford St, Cambridge MA 02138 phone: +1 617-496-5308 fax: +1 617-495-5667 www.oeb.harvard.edu/faculty/giribet/ Department of Marine Ecology Center for Advanced Studies of Blanes (CEAB-CSIC) C/Accés Cala St. Francesc 14 17300 Blanes, Girona, SPAIN phone: +34 972 336 101 fax: +34 972 337 806 www.ceab.csic.es On Fri, May 9, 2014 at 2:43 PM, Yuan Hao <yuan.x.hao@gmail.com> wrote: > Hi Alicia, > > Here is my two cents. I think RSEM isoform counts contain ‘multi- isoform’ > reads, i.e. reads mapped to multiple isoforms, but from a single gene, so I > don’t think DESeq2 would demonstrate it’s power in terms of accuracy in > your case. If your purpose is only to obtain an ‘inspiration of biology’ > from your data, why not simply use the fold change of your isoform > expression and to check out what’s the most changed ones? Yes, you probably > need some kind of normalization to leverage the library sizes. > > Cheers, > Yuan > > > On May 9, 2014, at 2:08 PM, Alicia R. Pérez-Porro < > alicia.r.perezporro@gmail.com> wrote: > > > Hi everyone, > > > > Yes, sorry Mike, I forgot to mention that I calculated my isoform counts > > estimation with RSEM. > > > > Thomas, I am aware about the pipeline using RSEM+(edgeR or DESeq) to do > DE > > analysis inside Trinity. I tried and I didn't like just because is not a > > good option to use with my data. I have non standard data and the > pipeline > > is too hands off so works nicely if you have more standard data. > > > > Mainly what I want is to do pair-wise comparisons between my 5 different > > conditions (5 different moments along the life cycle of my animal) to > > generate a list of the most DEG, blast them to see what they are, > > potentially get the GO:terms associated to them and have an overview of > > what is happening along the life cycle. My intention in the future is > > probably continue with analysis in differential exon expression (DEXseq) > > and/or differential isoform expression. And I am also considering > > time-scale differential expression analysis. But like I said I'm planning > > on doing that in the future because right now I am interested just in an > > overview of what is going on in the life cycle of my animal. That's why I > > was thinking about treating the isoforms as genes. > > > > Does that make sense to you, guys? > > > > Thanks again, > > Alicia > > > > > > > > -- > > Alicia R. Pérez-Porro > > PhD candidate > > > > Giribet lab > > Department of Organismic and Evolutionary Biology > > MCZ labs > > Harvard University > > 26 Oxford St, Cambridge MA 02138 > > phone: +1 617-496-5308 > > fax: +1 617-495-5667 > > www.oeb.harvard.edu/faculty/giribet/ > > > > Department of Marine Ecology > > Center for Advanced Studies of Blanes (CEAB-CSIC) > > C/Accés Cala St. Francesc 14 > > 17300 Blanes, Girona, SPAIN > > phone: +34 972 336 101 > > fax: +34 972 337 806 > > www.ceab.csic.es > > > > > > On Fri, May 9, 2014 at 1:45 PM, Michael Love < > michaelisaiahlove@gmail.com>wrote: > > > >> hi Alicia, > >> > >> In your previous email, you didn't mention that you had used software > >> for estimating isoform specific counts, so I pasted a link to Simon's > >> answer concerning isoform counts. If one can assign reads to isoforms > >> with high confidence then it would be fine to do testing with DESeq2. > >> However, this is typically a difficult task which involves uncertainty > >> in estimation and DESeq2 does not take into account any uncertainty in > >> these estimated counts. If you have 10 reads in the count matrix, > >> DESeq2 will take this to mean 10 reads were unambiguously assigned to > >> this feature. Through estimation steps, this could be 10 reads plus or > >> minus 10 reads. So the quality of the results will depend on the > >> quality of the input. > >> > >> Mike > >> > >> On Fri, May 9, 2014 at 11:11 AM, Alicia R. Pérez-Porro > >> <alicia.r.perezporro@gmail.com> wrote: > >>> Hi Mike, > >>> > >>> Thanks for your answer. I read the explanation from Simon and am still > a > >>> little bit confuse, reading the edgeR manual I found this: "edgeR can > be > >>> applied to di fferential expression at the gene, exon, transcript or > tag > >>> level. In fact, read counts can be summarized by any genomic feature. > >> edgeR > >>> analyses at the exon level are easily extended to detect di fferential > >>> splicing or isoform-specifi c dif ferential expression". > >>> > >>> So, my initial idea was to treat each isoform like a gene because for > the > >>> approach that I am attempting right now I am not interested in > >> differences > >>> in expression of isoforms belonging to the same gene. I did my de novo > >> with > >>> Trinity, alignment using Bowtie and estimation of abundance with RSEM. > I > >>> created a matrix with the RSEM counts (isoforms results) and I was > >> planning > >>> on using it as input for my next step. > >>> > >>> I understand that even I want to treat each isoform as a gene to do it > >> with > >>> DESeq or DESeq2 is not a good idea. I really don't know about edgeR and > >> am > >>> also considering EBseq. > >>> > >>> Suggestions? Thoughts? > >>> > >>> Thank you in advance. > >>> Alicia > >>> > >>> > >>> > >>> -- > >>> Alicia R. Pérez-Porro > >>> PhD candidate > >>> > >>> Giribet lab > >>> Department of Organismic and Evolutionary Biology > >>> MCZ labs > >>> Harvard University > >>> 26 Oxford St, Cambridge MA 02138 > >>> phone: +1 617-496-5308 > >>> fax: +1 617-495-5667 > >>> www.oeb.harvard.edu/faculty/giribet/ > >>> > >>> Department of Marine Ecology > >>> Center for Advanced Studies of Blanes (CEAB-CSIC) > >>> C/Accés Cala St. Francesc 14 > >>> 17300 Blanes, Girona, SPAIN > >>> phone: +34 972 336 101 > >>> fax: +34 972 337 806 > >>> www.ceab.csic.es > >>> > >>> > >>> On Fri, May 9, 2014 at 8:07 AM, Michael Love < > >> michaelisaiahlove@gmail.com> > >>> wrote: > >>>> > >>>> hi Alicia, > >>>> > >>>> DESeq2 is intended for gene-level analysis. here is some explanation > >> from > >>>> Simon on the list as to why doing a count-based analysis at the > >>>> transcript/isoform level is problematic: > >>>> > >>>> https://stat.ethz.ch/pipermail/bioconductor/2012-February/043410.html > >>>> > >>>> if you are interested in looking for differential exon usage, you can > >>>> instead consider using the DEXSeq package: > >>>> > >>>> publication > >>>> http://www.ncbi.nlm.nih.gov/pubmed/22722343 > >>>> > >>>> software: > >>>> http://bioconductor.org/packages/release/bioc/html/DEXSeq.html > >>>> > >>>> Mike > >>>> > >>>> > >>>> > >>>> On Thu, May 8, 2014 at 7:26 PM, Alicia R. Pérez-Porro > >>>> <alicia.r.perezporro@gmail.com> wrote: > >>>>> > >>>>> Dear Bioconductor users, > >>>>> > >>>>> I am working with differential expression at the transcript (isoform) > >>>>> level. I have 6 different conditions (2 replicates/condition). > >>>>> > >>>>> I want to know if I can use DESeq2 for that or if the package can > only > >> be > >>>>> used at the gene level. > >>>>> > >>>>> Thanks, > >>>>> Alicia > >>>>> > >>>>> > >>>>> -- > >>>>> Alicia R. Pérez-Porro > >>>>> PhD candidate > >>>>> > >>>>> Giribet lab > >>>>> Department of Organismic and Evolutionary Biology > >>>>> MCZ labs > >>>>> Harvard University > >>>>> 26 Oxford St, Cambridge MA 02138 > >>>>> phone: +1 617-496-5308 > >>>>> fax: +1 617-495-5667 > >>>>> www.oeb.harvard.edu/faculty/giribet/ > >>>>> > >>>>> Department of Marine Ecology > >>>>> Center for Advanced Studies of Blanes (CEAB-CSIC) > >>>>> C/Accés Cala St. Francesc 14 > >>>>> 17300 Blanes, Girona, SPAIN > >>>>> phone: +34 972 336 101 > >>>>> fax: +34 972 337 806 > >>>>> www.ceab.csic.es > >>>>> > >>>>> [[alternative HTML version deleted]] > >>>>> > >>>>> > >>>>> _______________________________________________ > >>>>> 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]] > > > > _______________________________________________ > > 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]]
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Thomas Girke ★ 1.7k
@thomas-girke-993
Last seen 9 months ago
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If you assembled your transcriptome, you might be aware that the Trinity site has some protocols for this that are based on RSEM and edgeR: http://trinityrnaseq.sourceforge.net/analysis/diff_expression_analysis .html Whether this is the best possible way of addressing this need is a different question. Thomas On Fri, May 09, 2014 at 04:38:54PM +0000, Alicia R. P?rez-Porro wrote: > Hi Xiayu, > > I don't think that I can use Cufflink, I don't have a reference genome, I > created my own reference transcriptome. > > Best, > Alicia > > > > -- > Alicia R. P?rez-Porro > PhD candidate > > Giribet lab > Department of Organismic and Evolutionary Biology > MCZ labs > Harvard University > 26 Oxford St, Cambridge MA 02138 > phone: +1 617-496-5308 > fax: +1 617-495-5667 > www.oeb.harvard.edu/faculty/giribet/ > > Department of Marine Ecology > Center for Advanced Studies of Blanes (CEAB-CSIC) > C/Acc?s Cala St. Francesc 14 > 17300 Blanes, Girona, SPAIN > phone: +34 972 336 101 > fax: +34 972 337 806 > www.ceab.csic.es > > > On Fri, May 9, 2014 at 12:09 PM, Rao,Xiayu <xrao at="" mdanderson.org=""> wrote: > > > Hi, Alicia > > > > You may try cufflinks. After running cuffdiff, you will get isoform.diff > > and other files, in which you will find the isoform expression level. > > Correct me if I am wrong. > > > > Thanks, > > Xiayu > > > > > > > > -----Original Message----- > > From: bioconductor-bounces at r-project.org [mailto: > > bioconductor-bounces at r-project.org] On Behalf Of Alicia R. P ?rez-Porro > > Sent: Thursday, May 08, 2014 6:26 PM > > To: bioconductor at r-project.org list > > Subject: [BioC] DESeq2 transcript level > > > > Dear Bioconductor users, > > > > I am working with differential expression at the transcript (isoform) > > level. I have 6 different conditions (2 replicates/condition). > > > > I want to know if I can use DESeq2 for that or if the package can only be > > used at the gene level. > > > > Thanks, > > Alicia > > > > > > -- > > Alicia R. P??rez-Porro > > PhD candidate > > > > Giribet lab > > Department of Organismic and Evolutionary Biology MCZ labs Harvard > > University > > 26 Oxford St, Cambridge MA 02138 > > phone: +1 617-496-5308 > > fax: +1 617-495-5667 > > www.oeb.harvard.edu/faculty/giribet/ > > > > Department of Marine Ecology > > Center for Advanced Studies of Blanes (CEAB-CSIC) C/Acc??s Cala St. > > Francesc 14 > > 17300 Blanes, Girona, SPAIN > > phone: +34 972 336 101 > > fax: +34 972 337 806 > > www.ceab.csic.es > > > > [[alternative HTML version deleted]] > > > > > > [[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
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Rao,Xiayu ▴ 550
@raoxiayu-6003
Last seen 9.5 years ago
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Hi, Alicia You may try cufflinks. After running cuffdiff, you will get isoform.diff and other files, in which you will find the isoform expression level. Correct me if I am wrong. Thanks, Xiayu -----Original Message----- From: bioconductor-bounces@r-project.org [mailto:bioconductor- bounces@r-project.org] On Behalf Of Alicia R. P?rez-Porro Sent: Thursday, May 08, 2014 6:26 PM To: bioconductor at r-project.org list Subject: [BioC] DESeq2 transcript level Dear Bioconductor users, I am working with differential expression at the transcript (isoform) level. I have 6 different conditions (2 replicates/condition). I want to know if I can use DESeq2 for that or if the package can only be used at the gene level. Thanks, Alicia -- Alicia R. P??rez-Porro PhD candidate Giribet lab Department of Organismic and Evolutionary Biology MCZ labs Harvard University 26 Oxford St, Cambridge MA 02138 phone: +1 617-496-5308 fax: +1 617-495-5667 www.oeb.harvard.edu/faculty/giribet/ Department of Marine Ecology Center for Advanced Studies of Blanes (CEAB-CSIC) C/Acc??s Cala St. Francesc 14 17300 Blanes, Girona, SPAIN phone: +34 972 336 101 fax: +34 972 337 806 www.ceab.csic.es [[alternative HTML version deleted]]
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Hi Xiayu, I don't think that I can use Cufflink, I don't have a reference genome, I created my own reference transcriptome. Best, Alicia -- Alicia R. Pérez-Porro PhD candidate Giribet lab Department of Organismic and Evolutionary Biology MCZ labs Harvard University 26 Oxford St, Cambridge MA 02138 phone: +1 617-496-5308 fax: +1 617-495-5667 www.oeb.harvard.edu/faculty/giribet/ Department of Marine Ecology Center for Advanced Studies of Blanes (CEAB-CSIC) C/Accés Cala St. Francesc 14 17300 Blanes, Girona, SPAIN phone: +34 972 336 101 fax: +34 972 337 806 www.ceab.csic.es On Fri, May 9, 2014 at 12:09 PM, Rao,Xiayu <xrao@mdanderson.org> wrote: > Hi, Alicia > > You may try cufflinks. After running cuffdiff, you will get isoform.diff > and other files, in which you will find the isoform expression level. > Correct me if I am wrong. > > Thanks, > Xiayu > > > > -----Original Message----- > From: bioconductor-bounces@r-project.org [mailto: > bioconductor-bounces@r-project.org] On Behalf Of Alicia R. Pérez- Porro > Sent: Thursday, May 08, 2014 6:26 PM > To: bioconductor@r-project.org list > Subject: [BioC] DESeq2 transcript level > > Dear Bioconductor users, > > I am working with differential expression at the transcript (isoform) > level. I have 6 different conditions (2 replicates/condition). > > I want to know if I can use DESeq2 for that or if the package can only be > used at the gene level. > > Thanks, > Alicia > > > -- > Alicia R. Pérez-Porro > PhD candidate > > Giribet lab > Department of Organismic and Evolutionary Biology MCZ labs Harvard > University > 26 Oxford St, Cambridge MA 02138 > phone: +1 617-496-5308 > fax: +1 617-495-5667 > www.oeb.harvard.edu/faculty/giribet/ > > Department of Marine Ecology > Center for Advanced Studies of Blanes (CEAB-CSIC) C/Accés Cala St. > Francesc 14 > 17300 Blanes, Girona, SPAIN > phone: +34 972 336 101 > fax: +34 972 337 806 > www.ceab.csic.es > > [[alternative HTML version deleted]] > > [[alternative HTML version deleted]]
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