Agilent arrays 2- or 1-color?
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Assa Yeroslaviz ★ 1.5k
@assa-yeroslaviz-1597
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Germany
Hi everybody, I have a data set of Agilent CGHArray. I would like to know whether these arrays were made with the two-chanel or one-channel protocol. When I look at the results from the Agilent FE files, I can see both g- and r-signals (eg. g(r)BGSubSignal, g(r)Median etc.) Asking the biologists I was told, that they are interested in the gBGSubSignal column of the results file. What I understand from that is, that the experiments ware made as single-channel exp. Is there something like that - a one-color experiment with a 2-color hybridization protocol? What kind of data do one has on the red channel, if this is really is a 1-channel experiment? As they also did one array with control (unchanged cell lines), I really think this is 1-channel arrays. Do you need a separate control experiments for a two-color arrays? Is it possible, that the red channel is some kind of a reference independent of the cell lines? I would appreciate the help Thanks Assa
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@sean-davis-490
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On Mon, Jul 30, 2012 at 2:49 PM, Assa Yeroslaviz <frymor@gmail.com> wrote: > Hi everybody, > > I have a data set of Agilent CGHArray. I would like to know whether > these arrays were made with the two-chanel or one-channel protocol. > When I look at the results from the Agilent FE files, I can see both > g- and r-signals (eg. g(r)BGSubSignal, g(r)Median etc.) > Asking the biologists I was told, that they are interested in the > gBGSubSignal column of the results file. What I understand from that > is, that the experiments ware made as single-channel exp. > Is there something like that - a one-color experiment with a 2-color > hybridization protocol? > > What kind of data do one has on the red channel, if this is really is > a 1-channel experiment? > > As they also did one array with control (unchanged cell lines), I > really think this is 1-channel arrays. Do you need a separate control > experiments for a two-color arrays? > Is it possible, that the red channel is some kind of a reference > independent of the cell lines? > > Hi, Assa. Your best bet is to sit down with the biologists who designed and ran the experiment to describe the experiment and the protocols used to produce the final data. Only then can you really understand what they want and how best to get that to them. Sean [[alternative HTML version deleted]]
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Hi, Sean, yes I know that. Unfortunately the biologist is not exactly next to me, as it is a collaboration. Well, I asked for the protocol. maybe I can figure it out. What I would like to know is, whether it is possible to do a two- channel hybridization protocol with just one biological probe on the array. Does it make sense at all? If so, what happened in the 'second' channel? Do I need to take both channel under consideration? If there is only one probe, will it spread onto both channels? thanks again, Assa On Mon, Jul 30, 2012 at 9:04 PM, Sean Davis <sdavis2@mail.nih.gov> wrote: > > > On Mon, Jul 30, 2012 at 2:49 PM, Assa Yeroslaviz <frymor@gmail.com> wrote: > >> Hi everybody, >> >> I have a data set of Agilent CGHArray. I would like to know whether >> these arrays were made with the two-chanel or one-channel protocol. >> When I look at the results from the Agilent FE files, I can see both >> g- and r-signals (eg. g(r)BGSubSignal, g(r)Median etc.) >> Asking the biologists I was told, that they are interested in the >> gBGSubSignal column of the results file. What I understand from that >> is, that the experiments ware made as single-channel exp. >> Is there something like that - a one-color experiment with a 2-color >> hybridization protocol? >> >> What kind of data do one has on the red channel, if this is really is >> a 1-channel experiment? >> >> As they also did one array with control (unchanged cell lines), I >> really think this is 1-channel arrays. Do you need a separate control >> experiments for a two-color arrays? >> Is it possible, that the red channel is some kind of a reference >> independent of the cell lines? >> >> > Hi, Assa. > > Your best bet is to sit down with the biologists who designed and ran the > experiment to describe the experiment and the protocols used to produce the > final data. Only then can you really understand what they want and how > best to get that to them. > > Sean > > > [[alternative HTML version deleted]]
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On Mon, Jul 30, 2012 at 10:55 PM, Assa Yeroslaviz <frymor@gmail.com> wrote: > Hi, Sean, > > yes I know that. Unfortunately the biologist is not exactly next to me, as > it is a collaboration. > A lot of us work on collaborations like this. > Well, I asked for the protocol. maybe I can figure it out. > > What I would like to know is, whether it is possible to do a two- channel > hybridization protocol with just one biological probe on the array. Does it > make sense at all? > > There are four possibilities, at least: 1) Two color hyb, two color scan 2) Two color hyb, one color scan 3) One color hyb, two color scan 4) One color hyb, one color scan It sounds like your data have both colors in the output file, so either 1 or 3 was done. > If so, what happened in the 'second' channel? Do I need to take both > channel under consideration? > This is the question you need to turn back to your biological collaborator. We cannot answer this question, unfortunately. > > If there is only one probe, will it spread onto both channels? > > The two channels are from the same probe. As I mentioned above, if you have two channels worth of data, there are at least two explanations. > thanks again, > > Assa > > On Mon, Jul 30, 2012 at 9:04 PM, Sean Davis <sdavis2@mail.nih.gov> wrote: > > > > > > > On Mon, Jul 30, 2012 at 2:49 PM, Assa Yeroslaviz <frymor@gmail.com> > wrote: > > > >> Hi everybody, > >> > >> I have a data set of Agilent CGHArray. I would like to know whether > >> these arrays were made with the two-chanel or one-channel protocol. > >> When I look at the results from the Agilent FE files, I can see both > >> g- and r-signals (eg. g(r)BGSubSignal, g(r)Median etc.) > >> Asking the biologists I was told, that they are interested in the > >> gBGSubSignal column of the results file. What I understand from that > >> is, that the experiments ware made as single-channel exp. > >> Is there something like that - a one-color experiment with a 2-color > >> hybridization protocol? > >> > >> What kind of data do one has on the red channel, if this is really is > >> a 1-channel experiment? > >> > >> As they also did one array with control (unchanged cell lines), I > >> really think this is 1-channel arrays. Do you need a separate control > >> experiments for a two-color arrays? > >> Is it possible, that the red channel is some kind of a reference > >> independent of the cell lines? > >> > >> > > Hi, Assa. > > > > Your best bet is to sit down with the biologists who designed and ran the > > experiment to describe the experiment and the protocols used to produce > the > > final data. Only then can you really understand what they want and how > > best to get that to them. > > > > Sean > > > > > > > > [[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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Hallo again, thanks for the help. I have found out that the experiment was done with a common reference. So if i understand it correctly I have here the first option you mentioned. so I gues my design matrix should looks like that: wt c1 c2 c3 c4 c5 c6 1 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 1 and my contrast.matrix something like that wt c1 c2 c3 c4 c5 c6 -1 1 0 0 0 0 0 -1 0 1 0 0 0 0 0 -1 0 1 0 0 0 0 -1 0 0 1 0 0 0 0 -1 0 0 1 0 0 0 -1 0 0 0 1 if i want to compare the first two with the control, number c3 & c4 with c1 and number c5 and c6 with c2 Do I need to also do a within-array normalization on this data set? Thanks Assa On Tue, Jul 31, 2012 at 12:51 PM, Sean Davis <sdavis2@mail.nih.gov> wrote: > > > On Mon, Jul 30, 2012 at 10:55 PM, Assa Yeroslaviz <frymor@gmail.com>wrote: > >> Hi, Sean, >> >> yes I know that. Unfortunately the biologist is not exactly next to me, as >> it is a collaboration. >> > > A lot of us work on collaborations like this. > > >> Well, I asked for the protocol. maybe I can figure it out. >> >> What I would like to know is, whether it is possible to do a two- channel >> hybridization protocol with just one biological probe on the array. Does >> it >> make sense at all? >> >> > There are four possibilities, at least: > > 1) Two color hyb, two color scan > 2) Two color hyb, one color scan > 3) One color hyb, two color scan > 4) One color hyb, one color scan > > It sounds like your data have both colors in the output file, so either 1 > or 3 was done. > > >> If so, what happened in the 'second' channel? Do I need to take both >> channel under consideration? >> > > This is the question you need to turn back to your biological > collaborator. We cannot answer this question, unfortunately. > > >> >> If there is only one probe, will it spread onto both channels? >> >> > The two channels are from the same probe. As I mentioned above, if you > have two channels worth of data, there are at least two explanations. > > >> thanks again, >> >> Assa >> >> On Mon, Jul 30, 2012 at 9:04 PM, Sean Davis <sdavis2@mail.nih.gov> wrote: >> >> > >> > >> > On Mon, Jul 30, 2012 at 2:49 PM, Assa Yeroslaviz <frymor@gmail.com> >> wrote: >> > >> >> Hi everybody, >> >> >> >> I have a data set of Agilent CGHArray. I would like to know whether >> >> these arrays were made with the two-chanel or one-channel protocol. >> >> When I look at the results from the Agilent FE files, I can see both >> >> g- and r-signals (eg. g(r)BGSubSignal, g(r)Median etc.) >> >> Asking the biologists I was told, that they are interested in the >> >> gBGSubSignal column of the results file. What I understand from that >> >> is, that the experiments ware made as single-channel exp. >> >> Is there something like that - a one-color experiment with a 2-color >> >> hybridization protocol? >> >> >> >> What kind of data do one has on the red channel, if this is really is >> >> a 1-channel experiment? >> >> >> >> As they also did one array with control (unchanged cell lines), I >> >> really think this is 1-channel arrays. Do you need a separate control >> >> experiments for a two-color arrays? >> >> Is it possible, that the red channel is some kind of a reference >> >> independent of the cell lines? >> >> >> >> >> > Hi, Assa. >> > >> > Your best bet is to sit down with the biologists who designed and ran >> the >> > experiment to describe the experiment and the protocols used to produce >> the >> > final data. Only then can you really understand what they want and how >> > best to get that to them. >> > >> > Sean >> > >> > >> > >> >> [[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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On Tue, Jul 31, 2012 at 8:54 AM, Assa Yeroslaviz <frymor@gmail.com> wrote: > Hallo again, > > thanks for the help. I have found out that the experiment was done with a > common reference. So if i understand it correctly I have here the first > option you mentioned. > > so I gues my design matrix should looks like that: > > wt c1 c2 c3 c4 c5 c6 > 1 0 0 0 0 0 > 0 1 0 0 0 0 > 0 0 1 0 0 0 > 0 0 0 1 0 0 > 0 0 0 0 1 0 > 0 0 0 0 0 1 > > and my contrast.matrix something like that > > wt c1 c2 c3 c4 c5 c6 > -1 1 0 0 0 0 0 > -1 0 1 0 0 0 0 > 0 -1 0 1 0 0 0 > 0 -1 0 0 1 0 0 > 0 0 -1 0 0 1 0 > 0 0 -1 0 0 0 1 > > if i want to compare the first two with the control, number c3 & c4 with c1 > and number c5 and c6 with c2 > > Hi, Assa. You can treat common-reference designs as single-color for the purposes of design and contrast matrices, so the limma user manual will be helpful in this regard. > Do I need to also do a within-array normalization on this data set? > > I would suggest doing within-array normalization, yes. You have options to deal with single channels, also. Again, see the limma user guide for details. Sean > Thanks > Assa > > On Tue, Jul 31, 2012 at 12:51 PM, Sean Davis <sdavis2@mail.nih.gov> wrote: > > > > > > > On Mon, Jul 30, 2012 at 10:55 PM, Assa Yeroslaviz <frymor@gmail.com> >wrote: > > > >> Hi, Sean, > >> > >> yes I know that. Unfortunately the biologist is not exactly next to me, > as > >> it is a collaboration. > >> > > > > A lot of us work on collaborations like this. > > > > > >> Well, I asked for the protocol. maybe I can figure it out. > >> > >> What I would like to know is, whether it is possible to do a two- channel > >> hybridization protocol with just one biological probe on the array. Does > >> it > >> make sense at all? > >> > >> > > There are four possibilities, at least: > > > > 1) Two color hyb, two color scan > > 2) Two color hyb, one color scan > > 3) One color hyb, two color scan > > 4) One color hyb, one color scan > > > > It sounds like your data have both colors in the output file, so either 1 > > or 3 was done. > > > > > >> If so, what happened in the 'second' channel? Do I need to take both > >> channel under consideration? > >> > > > > This is the question you need to turn back to your biological > > collaborator. We cannot answer this question, unfortunately. > > > > > >> > >> If there is only one probe, will it spread onto both channels? > >> > >> > > The two channels are from the same probe. As I mentioned above, if you > > have two channels worth of data, there are at least two explanations. > > > > > >> thanks again, > >> > >> Assa > >> > >> On Mon, Jul 30, 2012 at 9:04 PM, Sean Davis <sdavis2@mail.nih.gov> > wrote: > >> > >> > > >> > > >> > On Mon, Jul 30, 2012 at 2:49 PM, Assa Yeroslaviz <frymor@gmail.com> > >> wrote: > >> > > >> >> Hi everybody, > >> >> > >> >> I have a data set of Agilent CGHArray. I would like to know whether > >> >> these arrays were made with the two-chanel or one-channel protocol. > >> >> When I look at the results from the Agilent FE files, I can see both > >> >> g- and r-signals (eg. g(r)BGSubSignal, g(r)Median etc.) > >> >> Asking the biologists I was told, that they are interested in the > >> >> gBGSubSignal column of the results file. What I understand from that > >> >> is, that the experiments ware made as single-channel exp. > >> >> Is there something like that - a one-color experiment with a 2-color > >> >> hybridization protocol? > >> >> > >> >> What kind of data do one has on the red channel, if this is really is > >> >> a 1-channel experiment? > >> >> > >> >> As they also did one array with control (unchanged cell lines), I > >> >> really think this is 1-channel arrays. Do you need a separate control > >> >> experiments for a two-color arrays? > >> >> Is it possible, that the red channel is some kind of a reference > >> >> independent of the cell lines? > >> >> > >> >> > >> > Hi, Assa. > >> > > >> > Your best bet is to sit down with the biologists who designed and ran > >> the > >> > experiment to describe the experiment and the protocols used to > produce > >> the > >> > final data. Only then can you really understand what they want and > how > >> > best to get that to them. > >> > > >> > Sean > >> > > >> > > >> > > >> > >> [[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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Hi again, ok I have found out, that there is really two biological probes on all arrays. There is a common reference on all of them. If I have a common reference for all the arrays, can I use the arrays as a one-channel experiment? Do I ignore the second channel? What do I need the second channel for? Thanks Assa On Tue, Jul 31, 2012 at 7:55 AM, Assa Yeroslaviz <frymor@gmail.com> wrote: > Hi, Sean, > > yes I know that. Unfortunately the biologist is not exactly next to me, as > it is a collaboration. > Well, I asked for the protocol. maybe I can figure it out. > > What I would like to know is, whether it is possible to do a two- channel > hybridization protocol with just one biological probe on the array. Does it > make sense at all? > > If so, what happened in the 'second' channel? Do I need to take both > channel under consideration? > > If there is only one probe, will it spread onto both channels? > > thanks again, > > Assa > > > On Mon, Jul 30, 2012 at 9:04 PM, Sean Davis <sdavis2@mail.nih.gov> wrote: > >> >> >> On Mon, Jul 30, 2012 at 2:49 PM, Assa Yeroslaviz <frymor@gmail.com>wrote: >> >>> Hi everybody, >>> >>> I have a data set of Agilent CGHArray. I would like to know whether >>> these arrays were made with the two-chanel or one-channel protocol. >>> When I look at the results from the Agilent FE files, I can see both >>> g- and r-signals (eg. g(r)BGSubSignal, g(r)Median etc.) >>> Asking the biologists I was told, that they are interested in the >>> gBGSubSignal column of the results file. What I understand from that >>> is, that the experiments ware made as single-channel exp. >>> Is there something like that - a one-color experiment with a 2-color >>> hybridization protocol? >>> >>> What kind of data do one has on the red channel, if this is really is >>> a 1-channel experiment? >>> >>> As they also did one array with control (unchanged cell lines), I >>> really think this is 1-channel arrays. Do you need a separate control >>> experiments for a two-color arrays? >>> Is it possible, that the red channel is some kind of a reference >>> independent of the cell lines? >>> >>> >> Hi, Assa. >> >> Your best bet is to sit down with the biologists who designed and ran the >> experiment to describe the experiment and the protocols used to produce the >> final data. Only then can you really understand what they want and how >> best to get that to them. >> >> Sean >> >> >> > > [[alternative HTML version deleted]]
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