Thank you for the reply. I actually need to get the log2 copy number ratios form the raw .cel files of a GenomeWideSNP6.0 array - I was using CRMA1 but am now repeating the analysis using CRMA v2. I am putting all of the different tumour types either matched or unmatched with a germline sample in the same directory and all the normal samples in another directory. I will then go through the processes of qulaity assesment, calibration crosstalk, normalization for probe sequence effect, probe summarization, and normalization of PCR fragment length effects.
Do I need to calculate the raw copy numbers and turn these into log2 copy numbers? How would I then calculate the copy numbers for 1. Unpaired tumour samples - will need to be compared to a pooled reference from a particular tumpur type 2. Paired samples? Many thanks for your help On 18/07/2010 12:01, "Ajanthah Sangaralingam" <a.sangaralin...@qmul.ac.uk> wrote: > Hi, > > Yes this is correct. > > Many thanks > > Ajanthah > ________________________________________ > From: aroma-affymetrix@googlegroups.com [aroma-affymet...@googlegroups.com] On > Behalf Of Henrik Bengtsson [...@stat.berkeley.edu] > Sent: Sunday, July 18, 2010 11:28 AM > To: aroma-affymetrix > Subject: Re: [aroma.affymetrix] Analysis of GenomeWideSNP6.0 data > > Hi. > > On Fri, Jul 16, 2010 at 11:13 AM, Ajanthah Sangaralingam > <a.sangaralin...@qmul.ac.uk> wrote: >> Hi, >> >> I have been doing some paired total copy number analysis in aroma afyymetrix. >> The dataset I have is complicated for haf the dataset I have reference >> samples, for the other half I will do an unpiared analysis. > > So, to make sure I don't misunderstand, you have an Affymetrix > GenomeWideSNP_6 (GWS6) data set that contains tumors and for some, but > not all of the you have matched normal samples, where "matched normal" > mean a normal tissue or normal blood extract from the same patient as > the tumor was taken. Is this correct? > >> I alos have data from many different tomor types not just one - I do not have >> the sample number of samples from each type of tumor. >> >> My questions are: >> >> When doing a paired analysis - the normal and tumour data have there own >> directories and allelic cross talk calibration, summarization and PCR >> fragment length normlization is all done separately. > > It is important to know which preprocessing method you are following. > Since you are working with GWS6 arrays, I recommend that you use the > CRMAv2 preprocessing method as described in vignette 'Estimation of > total copy numbers using the CRMA v2 method (10K-GWS6)': > > http://aroma-project.org/vignettes/CRMAv2 > > Note the function doCRMAv2() which is convenient when you do not want > to dig into the details. > > Since you are not mentioning probe-sequence normalization, it looks > like you are indeed using CRMA v1. If so, I recommend that you use > CRMA v2 instead. Using CRMA v2 will be really useful for you, as > explained below. > >> Is this tue for the different tumor types as well - should they be treated >> separately for all of tehse stages or can all the tumor types be put into one >> tumour directory. > > This is perfectly fine if you are using CRMA v2 (but not CRMA v1). > As now clarified in the vignette, in addition to the CRMAv2 paper, you > will get identical results with CRMAv2 regardless what other samples > you put in your data set; the CRMAv2 method is truly a single-array > method. It is only when you get to the step where calculate copy > numbers relative to a pool of references you have to make a decision > on what pool of reference samples you'll use. > >> Also, I am unable to extarct the reference samples that I want after >> normaization to compare to the matching sanmples say in another tumor type. >> Segmentation models cannot be fit unless the number of samples match exactly. > > It actually can, as explained below. > >> >> Does this mean that I need to do all the stages again for the subsets of >> reference samples that have matching pairs in the other tumor types? > > The segmentation models, for instance CbsModel, segments each tumor > either (a) to a matched normal, or (b) to a global reference. When > you do (a), by definition there has to be an equal number of tumors as > matched normals, whereas when you do (b), there can only be one > reference sample specified. > > Example of paired tumor-normal segmentation: > > # A set of tumor samples > dsT <- ... > # A set of matched normal samples ordered such that they > # match the ordering in the tumor data set 'dsT'. > dsN <- ... > sm <- CbsModel(dsT, dsN); > > Example of tumor-global reference segmentation: > > # A set of tumor samples > dsT <- ... > # A set of reference samples (can be normals or everything) > dsR <- ... > # Use the pool of all reference samples as the reference > dfR <- getAverageFile(dsR); > sm <- CbsModel(dsT, dfR); > > Note that 'dfR' is a single "virtual" array, not a data set. > > Did that above make sense? > > /Henrik > >> >> Many thanks >> >> Ajanthah >> >> >> >> >> This email may contain information that is privileged, confidential or >> otherwise protected from disclosure. >> It must not be used by, or its contents copied or disclosed to, persons other >> than the addressee. >> If you have received this email in error please notify the sender immediately >> and delete the email. >> This message has been scanned for viruses. >> >> -- >> When reporting problems on aroma.affymetrix, make sure 1) to run the latest >> version of the package, 2) to report the output of sessionInfo() and >> traceback(), and 3) to post a complete code example. >> >> >> You received this message because you are subscribed to the Google Groups >> "aroma.affymetrix" group with website http://www.aroma-project.org/. >> To post to this group, send email to aroma-affymetrix@googlegroups.com >> To unsubscribe and other options, go to http://www.aroma-project.org/forum/ >> > > -- > When reporting problems on aroma.affymetrix, make sure 1) to run the latest > version of the package, 2) to report the output of sessionInfo() and > traceback(), and 3) to post a complete code example. > > > You received this message because you are subscribed to the Google Groups > "aroma.affymetrix" group with website http://www.aroma-project.org/. > To post to this group, send email to aroma-affymetrix@googlegroups.com > To unsubscribe and other options, go to http://www.aroma-project.org/forum/ -- When reporting problems on aroma.affymetrix, make sure 1) to run the latest version of the package, 2) to report the output of sessionInfo() and traceback(), and 3) to post a complete code example. 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