hi all, a rookie tilingArray question. i'm having trouble creating the perfect match index vector. for my probe set (n= 6490), each probe is a unique, perfect match to its target. i have created a probe index file that contains the integers: 1, 2....6490 (one index per line). i scanned this file as: pm_vector <- scan("PerfectMatchIndex", 0) i used the pm_vector in a following command: vac_normalized_exp_data <- normalizeByReference(vac_exp_set, vac_hyb_set, pm= pm_vector, background= bg_vector, cutoffQuantile= 0) this gives me an error: 'pm' must be an integer vector with values between 1 and 6490. any idea what the problem could be? tia anjan -- =========================================== anjan purkayastha, phd bioinformatics analyst whitehead institute for biomedical research nine cambridge center cambridge, ma 02142 purkayas [at] wi [dot] mit [dot] edu 703.740.6939

Hello Anjan, well, the error message indicates that your vector pm_vector is not usable by the function. Is it really an integer vector. What is the output of str(pm_vector) ? Probably, it would be better to read in the file by something like pm_vector <- read.delim("PerfectMatchIndex", skip=<put number="" of="" header="" lines="" here!="">)$V1 Again, check the result using str(pm_vector) If you want to use other functionalities of package tilingArray, you should consider creating a 'probeAnno' environment, which holds the mapping between probes and genomic match positions, for your array. Please see the script "makeProbeAnno.R" in the package's "scripts" directory for details. Once you have such a probeAnno object, the function "PMindex" can be used to obtain a numeric vector that you can use as "pm" in the normalization function. Please see the vignette "assessNorm" of package tilingArray for more details. Best regards, Joern Anjan Purkayastha wrote: > hi all, > a rookie tilingArray question. > i'm having trouble creating the perfect match index vector. > for my probe set (n= 6490), each probe is a unique, perfect match to > its target. > i have created a probe index file that contains the integers: 1, > 2....6490 (one index per line). > i scanned this file as: pm_vector <- scan("PerfectMatchIndex", 0) > > i used the pm_vector in a following command: > vac_normalized_exp_data <- normalizeByReference(vac_exp_set, > vac_hyb_set, pm= pm_vector, background= bg_vector, cutoffQuantile= 0) > > this gives me an error: > 'pm' must be an integer vector with values between 1 and 6490. > > any idea what the problem could be? > tia > anjan > -- Joern Toedling EMBL - European Bioinformatics Institute Wellcome Trust Genome Campus Hinxton, Cambridge CB10 1SD United Kingdom Phone +44(0)1223 492566 Email toedling at ebi.ac.uk

Anjan Purkayastha wrote: > joern, > thanks. > let me modify a local version and see the results. is it ok if i work > on this problem with you? i think dr. huber's method to genomic dna > hybridization based approach suits our purpose the best. > to give you a brief idea about our platform: we have built a whole > genome tiling array for the vaccinia virus (ds DNA virus, genome size: > ca 190kb); there are about 19000 overlapping probes, 60 bp in length, > for each strand. for the purposes of normalization i have removed > overlapping probes and am left with 6488 non-overlapping, contiguous > members. > > anyway, i'll rerun the procedure with the revised script and see what > i come up with . > thank you for your help. > warm regards, > anjan > > > > > Joern Toedling wrote: >> Anjan, >> looking at the source code of the function normalizeByReference, there >> is indeed the line >> vsnMatrix(xn, lts.quantile = 0.95, subsample = as.integer(2e+05)) , >> and since your matrix only has 6488 lines, this cannot work. >> Without knowing more details about your array platform and study, I >> cannot say whether or not the function "normalizeByReference" is really >> applicable to your array platform. However, we should augment the >> function anyway by chaning this line to something like >> vsnMatrix(xn, lts.quantile = 0.95, subsample = min(nrow(xn), >> as.integer(2e+05))) . >> We are going to make this change and it will be included in the >> development version of tilingArray within a few days. For the moment, >> you will have to work with a thus modified local copy of the function. >> Sorry for the inconvenience. >> >> Regards, >> Joern >> >> >> Anjan Purkayastha wrote: >> >>> joern, >>> thanks. i've solved the problem by wrapping an as. integer function >>> around the pm_vector. so here is my command: >>> >>> vac_normalized_data= normalizeByReference(vac_exp_set, vac_hyb_set, >>> pm= as.integer(pm_vector), background= as.integer(bg_vector)) >>> >>> however my story does not end here. i get a further set of errors >>> after this command. >>> Error in validObject(.Object) : >>> invalid class "vsnInput" object: 'subsample' must be a numeric vector >>> of length 1 with values between 0 and 6488. >>> In addition: Warning message: >>> In normalizeByReference(vac_exp_set, vac_hyb_set, pm = >>> as.integer(pm_vector), : >>> 'some strata of background contain fewer than 5000 features, are you >>> sure this is alright? >>> Error in exprs(vsnMatrix(xn, lts.quantile = 0.95, subsample = >>> as.integer(2e+05), : >>> error in evaluating the argument 'object' in selecting a method for >>> function 'exprs' >>> >>> the first part of the error message: vsnInput" object: 'subsample' >>> must be a numeric vector of length 1 with values between 0 and 6488, >>> does not make much sense to me. >>> if i understand the second part of the error message about background >>> strata correctly, there are less than 5000 features in some background >>> strata. that is understandable as i have only 1275 probes in the >>> background set. there are a total of 6488 probes in my set. >>> >>> any ideas on how to troubleshoot this? >>> thanks. >>> anjan >>> >>> >>> >>> >> >> > > -- =========================================== anjan purkayastha, phd bioinformatics analyst whitehead institute for biomedical research nine cambridge center cambridge, ma 02142 purkayas [at] wi [dot] mit [dot] edu 703.740.6939