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 [[alternative HTML version deleted]]