Question: Normalisation of Agilent microRNA arrays using inbuilt controls?
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gravatar for Daniel Brewer
9.7 years ago by
Daniel Brewer1.9k
Daniel Brewer1.9k wrote:
Hello, I have been trying to work out the best way to normalise microRNA microarrays recently as there seems to be some debate about whether the normal mRNA assumptions hold. The AgiMicroRna suggests using quantile or scale, whereas other posts I have read have suggested VSN. My question is whether the control probes on the Agilent chip could be used o do the normalisation i.e. the Negative and Positive Control probes. Would this be a good idea? And how would one go about this? A loess using the weights to define what probes are used? Thanks Dan -- ************************************************************** Daniel Brewer, Ph.D. Institute of Cancer Research Molecular Carcinogenesis Email: daniel.brewer at icr.ac.uk ************************************************************** The Institute of Cancer Research: Royal Cancer Hospital, a charitable Company Limited by Guarantee, Registered in England under Company No. 534147 with its Registered Office at 123 Old Brompton Road, London SW7 3RP. This e-mail message is confidential and for use by the a...{{dropped:2}}
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ADD COMMENTlink modified 9.7 years ago by Wei Shi3.2k • written 9.7 years ago by Daniel Brewer1.9k
Answer: Normalisation of Agilent microRNA arrays using inbuilt controls?
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gravatar for Wei Shi
9.7 years ago by
Wei Shi3.2k
Australia
Wei Shi3.2k wrote:
Dear Daniel: You might need to check the quality of these negative controls to see if they provide a good measurement of the background noise. One way to do this is to use the density function to check if these probes are normally distributed. If the negative control probes are a good representation of the background noise, you can consider using these controls to estimate the background mean intensity and the standard deviation of the background intensities which are two of the parameters needed by the normexp background correction model. The limma function neqc uses the negative controls and positive controls to do the normalization which might be useful for your data. Cheers, Wei Daniel Brewer wrote: > Hello, > > I have been trying to work out the best way to normalise microRNA > microarrays recently as there seems to be some debate about whether the > normal mRNA assumptions hold. > > The AgiMicroRna suggests using quantile or scale, whereas other posts I > have read have suggested VSN. > > My question is whether the control probes on the Agilent chip could be > used o do the normalisation i.e. the Negative and Positive Control probes. > > Would this be a good idea? And how would one go about this? A loess > using the weights to define what probes are used? > > Thanks > > Dan > > ______________________________________________________________________ The information in this email is confidential and intend...{{dropped:4}}
ADD COMMENTlink written 9.7 years ago by Wei Shi3.2k
Hi, in AgiMicroRNa we don´t advocate for any particular method of normalization. We have included scale, quantile and RMA and we let the user decide what might best method for them. You may even want to try some other methods that are not currently implemented in the package. Recently we have published “Processing of Agilent microRNA array data” (López-Romero et al. BMC Research Notes 2010, 3:18) and we concluded that using the AFE signal + quantiles or using the Raw mean signal + RMA method (with not background) might be appropriate methods for the preprocessing of the microRNA signals. Although in that paper we focused mainly in the way that the replicated probes are summarized in to a single expression measure and we only assessed the methods in terms of the signal variability. According to Agilent the controls cannot be used for normalization because they don’t behave consistenly, but this is something that I haven´t checked myself. You can try what Wei suggests. p.- On Fri, Feb 12, 2010 at 12:02 AM, Wei Shi <shi@wehi.edu.au> wrote: > Dear Daniel: > > You might need to check the quality of these negative controls to see if > they provide a good measurement of the background noise. One way to do this > is to use the density function to check if these probes are normally > distributed. > > If the negative control probes are a good representation of the > background noise, you can consider using these controls to estimate the > background mean intensity and the standard deviation of the background > intensities which are two of the parameters needed by the normexp background > correction model. The limma function neqc uses the negative controls and > positive controls to do the normalization which might be useful for your > data. > > Cheers, > Wei > > > Daniel Brewer wrote: > >> Hello, >> >> I have been trying to work out the best way to normalise microRNA >> microarrays recently as there seems to be some debate about whether the >> normal mRNA assumptions hold. >> >> The AgiMicroRna suggests using quantile or scale, whereas other posts I >> have read have suggested VSN. >> >> My question is whether the control probes on the Agilent chip could be >> used o do the normalisation i.e. the Negative and Positive Control probes. >> >> Would this be a good idea? And how would one go about this? A loess >> using the weights to define what probes are used? >> >> Thanks >> >> Dan >> >> >> > > ______________________________________________________________________ > The information in this email is confidential and intend...{{dropped:4}} > > > _______________________________________________ > Bioconductor mailing list > Bioconductor@stat.math.ethz.ch > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: > http://news.gmane.org/gmane.science.biology.informatics.conductor > [[alternative HTML version deleted]]
ADD REPLYlink written 9.7 years ago by Pedro Lopez-Romero120
Could anybody please tell me what the input list (myPeakList) for the ChIPpeakAnno program look like? I have a list of probeset ids with genomic coordinates coming from a nimblegen 385k chip chip experiment. Thanks in advance, Som. _________________________________________________________________ Hotmail: Trusted email with Microsoft’s powerful SPAM protection. [[alternative HTML version deleted]]
ADD REPLYlink written 9.7 years ago by fire1976 wyoming380
Hi Som, myPeakList is RangedData, where "start" is the start (or summit) of the binding site, "end" is the end of the binding site, "names" is the name of the binding site and "space" is the chromosome name. Here is how to create RangedData myexp from a list of binding sites. myexp = RangedData(IRanges(start = c(967654, 2010897, 2496704), end = c(967754, 2010997, 2496804), names = c("Site1", "Site2", "Site3")), space = c("1", "2", "3")) Please see ?annotatePeakInBatch for more examples. Thanks! Best regards, Julie ******************************************* Lihua Julie Zhu, Ph.D Research Associate Professor Program Gene Function and Expression University of Massachusetts Medical School 364 Plantation Street, Room 613 Worcester, MA 01605 508-856-5256 http://www.umassmed.edu/pgfe/faculty/zhu.cfm On 2/19/10 8:52 AM, "somnath bandyopadhyay" <genome1976 at="" hotmail.com=""> wrote: > > Could anybody please tell me what the input list (myPeakList) for the > ChIPpeakAnno program look like? > > I have a list of probeset ids with genomic coordinates coming from a nimblegen > 385k chip chip experiment. > > > > Thanks in advance, > > Som. > > _________________________________________________________________ > Hotmail: Trusted email with Microsoft?s powerful SPAM protection. > > [[alternative HTML version deleted]] > > _______________________________________________ > Bioconductor mailing list > Bioconductor at stat.math.ethz.ch > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: > http://news.gmane.org/gmane.science.biology.informatics.conductor
ADD REPLYlink written 9.7 years ago by Julie Zhu4.0k
Is there a way to write the results from annotatePeakInBatch( ? Thanks in advance. Som. > Date: Fri, 19 Feb 2010 09:43:09 -0500 > Subject: Re: [BioC] ChIPpeakAnno > From: julie.zhu@umassmed.edu > To: genome1976@hotmail.com; bioconductor@stat.math.ethz.ch > > Hi Som, > > myPeakList is RangedData, where "start" is the start (or summit) of the > binding site, "end" is the end of the binding site, "names" is the name of > the binding site and "space" is the chromosome name. > > Here is how to create RangedData myexp from a list of binding sites. > > myexp = RangedData(IRanges(start = c(967654, 2010897, 2496704), end = > c(967754, 2010997, 2496804), names = c("Site1", "Site2", "Site3")), space = > c("1", "2", "3")) > > Please see ?annotatePeakInBatch for more examples. Thanks! > > Best regards, > > Julie > > > ******************************************* > Lihua Julie Zhu, Ph.D > Research Associate Professor > Program Gene Function and Expression > University of Massachusetts Medical School > 364 Plantation Street, Room 613 > Worcester, MA 01605 > 508-856-5256 > http://www.umassmed.edu/pgfe/faculty/zhu.cfm > > > > On 2/19/10 8:52 AM, "somnath bandyopadhyay" <genome1976@hotmail.com> wrote: > > > > > Could anybody please tell me what the input list (myPeakList) for the > > ChIPpeakAnno program look like? > > > > I have a list of probeset ids with genomic coordinates coming from a nimblegen > > 385k chip chip experiment. > > > > > > > > Thanks in advance, > > > > Som. > > > > _________________________________________________________________ > > Hotmail: Trusted email with Microsoft‚s powerful SPAM protection. > > > > [[alternative HTML version deleted]] > > > > _______________________________________________ > > Bioconductor mailing list > > Bioconductor@stat.math.ethz.ch > > https://stat.ethz.ch/mailman/listinfo/bioconductor > > Search the archives: > > http://news.gmane.org/gmane.science.biology.informatics.conductor > > _________________________________________________________________ Hotmail: Free, trusted and rich email service. [[alternative HTML version deleted]]
ADD REPLYlink written 9.7 years ago by fire1976 wyoming380
Hi Som, You can write the result to a file using write.table(as.data.frame(annotatedPeak), file=”yourfile.xls”, sep=”\t”, row.names=FALSE). The documentation at http://www.bioconductor.org/packages/bioc/vignettes/ChIPpeakAnno/inst/ doc/Ch IPpeakAnno.pdf might be useful if you have not looked at it yet. Best regards, Julie On 2/20/10 5:11 PM, "somnath bandyopadhyay" <genome1976@hotmail.com> wrote: > Is there a way to write the results from annotatePeakInBatch( ? > > Thanks in advance. > Som. > >> > Date: Fri, 19 Feb 2010 09:43:09 -0500 >> > Subject: Re: [BioC] ChIPpeakAnno >> > From: julie.zhu@umassmed.edu >> > To: genome1976@hotmail.com; bioconductor@stat.math.ethz.ch >> > >> > Hi Som, >> > >> > myPeakList is RangedData, where "start" is the start (or summit) of the >> > binding site, "end" is the end of the binding site, "names" is the name of >> > the binding site and "space" is the chromosome name. >> > >> > Here is how to create RangedData myexp from a list of binding sites. >> > >> > myexp = RangedData(IRanges(start = c(967654, 2010897, 2496704), end = >> > c(967754, 2010997, 2496804), names = c("Site1", "Site2", "Site3")), space = >> > c("1", "2", "3")) >> > >> > Please see ?annotatePeakInBatch for more examples. Thanks! >> > >> > Best regards, >> > >> > Julie >> > >> > >> > ******************************************* >> > Lihua Julie Zhu, Ph.D >> > Research Associate Professor >> > Program Gene Function and Expression >> > University of Massachusetts Medical School >> > 364 Plantation Street, Room 613 >> > Worcester, MA 01605 >> > 508-856-5256 >> > http://www.umassmed.edu/pgfe/faculty/zhu.cfm >> > >> > >> > >> > On 2/19/10 8:52 AM, "somnath bandyopadhyay" <genome1976@hotmail.com> wrote: >> > >>> > > >>> > > Could anybody please tell me what the input list (myPeakList) for the >>> > > ChIPpeakAnno program look like? >>> > > >>> > > I have a list of probeset ids with genomic coordinates coming from a >>> nimblegen >>> > > 385k chip chip experiment. >>> > > >>> > > >>> > > >>> > > Thanks in advance, >>> > > >>> > > Som. >>> > > >>> > > _________________________________________________________________ >>> > > Hotmail: Trusted email with Microsoft‚s powerful SPAM protection. >>> > > >>> > > [[alternative HTML version deleted]] >>> > > >>> > > _______________________________________________ >>> > > Bioconductor mailing list >>> > > Bioconductor@stat.math.ethz.ch >>> > > https://stat.ethz.ch/mailman/listinfo/bioconductor >>> > > Search the archives: >>> > > http://news.gmane.org/gmane.science.biology.informatics.conductor >> > >> > > > > Hotmail: Free, trusted and rich email service. Get it now. > <http: clk.atdmt.com="" gbl="" go="" 201469228="" direct="" 01=""/> [[alternative HTML version deleted]]
ADD REPLYlink written 9.7 years ago by Julie Zhu4.0k
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