Johnson, William Evan <wej at="" ...=""> writes: > > Hey Magda, > > The two-step method is still a reasonable approach. It has worked well for me in multiple situations. I do > have a beta version of a ComBat version that will handle two batch variables at the same time. It works well > in theory--but I have yet to test it thoroughly across multiple datasets. I'm willing to share the code if > you want to test it on your data (let me know). > > ComBat in the sva package can handle numeric covariates, but it does not deal with continuous batch > variables. Adjusting the mean of a continuous batch variable would be straight-forward (assuming a > linear effect), but the variance adjustment would be very tricky. > > Ultimately, since the two-step approach seems to have worked, I think your best option is to just move > forward with those results. > > Thanks! > > Evan > > On Feb 19, 2014, at 4:00 AM, <bioconductor-request at="" ...=""> > <bioconductor-request at="" ...=""> wrote: > > > Message: 23 > > Date: Tue, 18 Feb 2014 16:45:12 -0800 > > From: Magda Price <magdaprice at="" ...=""> > > To: "bioconductor at ..." <bioconductor at="" ...=""> > > Subject: [BioC] ComBat: 2 adjustment variables & continuous adjustment > > variables > > Message-ID: > > <cadkr4v=ydd1abjxfhtd+xwq8mzmp_=urhvdtpxoturpqjzb7tg at="" ...=""> > > Content-Type: text/plain > > > > Hi! > > > > I'm writing with a few questions about applying ComBat (sva package) to a > > set of ~50 samples run on the the Illumina Infinium HumanMethylation450 > > BeadChip array (~450,000 DNA methylation data points). > > > > There is a large amount of variation in my data due to both the batch the > > samples were run in (3 different batches), in addition to the position they > > were located on the chip - specifically the row (6 different rows), but not > > the column. The chips are set up in a 6 row * 2 column format like this: > > > > > > sample 01 sample 02 > > sample 03 sample 04 > > sample 05 sample 06 > > sample 07 sample 08 > > sample 09 sample 10 > > sample 11 sample 12 > > > > > > I read Dr. Evan Johnson's suggestions to someone else with this > > "2-batch-effect-variable" problem in the ComBat google group ( > > https://groups.google.com/forum/#!topic/combat-user- forum/PcTxNlaUmAI). He > > had 2 good suggestions: > > > > 1. Combine the two batch variables into one, if 3-4 reps are left in > > each batch > > 2. Use ComBat twice, adjusting for the first batch using the second > > batch as a covariate, and then adjust for the second batch. > > > > I cannot go with the first suggestion because combining the 2 batch > > variables would create 18 batch categories (3 batches * 6 rows), and I > > would not have enough replicates per batch category. > > > > So I tried the second option - applying ComBat twice. I first corrected for > > row and then took the row-corrected data and applied ComBat again, > > correcting for batch. It seems to have worked & the correlation of my > > technical replicates improves. I am seeking advice on two points: > > > > 1. The google group post is now a few years old, is it still thought > > that the step-wise correction is a valid approach? > > 2. Row would be better treated as a continuous adjustment variable than > > a factor. In the version of sva that I am using (3.0.2) I believe that only > > factors adjustment variables are supported. I have seen mention in a few > > forums that there might be an update to ComBat to adjust for a numeric > > batch variable, is one available? > > > > Thank you in advanced for your help! > > > > Magda Price, > > University of British Columbia > > _______________________________________________ > Bioconductor mailing list > Bioconductor at ... > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: http://news.gmane.org/gmane.science.biology.informatics.conductor > > Hi Evan, Thanks for your response & so sorry for my delay, I wasn't notified by e- mail that you had responded. Since I wrote the first note, a few things have changed: 1. I have discovered a third batch variable (chip, in addition to plate & row)! 2. Based on forum feedback, I figured I was okay to stick with all factor adjustment variables. An additional question that has come up in reference to your suggestion: > > Use ComBat twice, adjusting for the first batch using the second > > batch as a covariate, and then adjust for the second batch. I can't include the second batch variable as a covariate; I get a singularity error because the batches are confounded with each other. For example, all samples on chip A were run in batch 1. Do you still think it a valid approach if I can't use subsequent batch variables as covariates? Thank you for the offer & advice! Magda