Hello to all ComBat users and to Dr. Evan, I am trying to remove the batch effects from some of my microarray data but at last I am getting an error message which read as Found 3 batches Found 1 categorical covariate(s) Standardizing Data across genes Error in solve.default(t(design) %*% design) : Lapack routine dgesv: system is exactly singular: U[4,4] = 0 The head(edata) looks like this AL AO AP AQ CF GT_pfalci_specific_0000001 16.053898 16.080540 16.101114 16.046898 16.087206 GT_pfalci_specific_0000002 10.051407 10.477143 8.369233 10.657850 13.312936 GT_pfalci_specific_0000003 8.910620 8.683393 7.812817 8.496099 10.920685 GT_pfalci_specific_0000004 6.603195 8.993232 6.476777 6.792369 3.319346 GT_pfalci_specific_0000005 9.813562 11.084574 9.055613 11.568550 12.977261 GT_pfalci_specific_0000006 15.989252 15.993513 15.963054 16.000675 15.983985 CL CU CV GA_UC GB_UC GT_pfalci_specific_0000001 16.082037 16.071299 16.090370 15.971335 15.994304 GT_pfalci_specific_0000002 12.653076 9.703247 8.827624 5.697412 8.060719 GT_pfalci_specific_0000003 11.470758 10.548943 10.718349 6.132614 8.007271 GT_pfalci_specific_0000004 5.328515 8.398546 6.351136 3.045112 3.891578 GT_pfalci_specific_0000005 8.520699 11.791610 11.535907 6.791468 9.930246 GT_pfalci_specific_0000006 15.980660 15.984256 15.970124 13.353012 13.740395 GC_UC GE_UC GR_UC GT_pfalci_specific_0000001 15.855644 16.090246 16.086956 GT_pfalci_specific_0000002 9.026398 8.015609 7.814614 GT_pfalci_specific_0000003 5.341252 8.658231 5.788790 GT_pfalci_specific_0000004 4.191565 3.040515 3.517175 GT_pfalci_specific_0000005 5.446910 11.982848 5.477334 GT_pfalci_specific_0000006 11.872469 13.675290 13.117105 GT_pfalci_specific_0000006 15.983985 15.970124 and the head(pheno) looks like this sample batch malaria AL 1 1 severe AO 2 1 severe AP 3 1 severe AQ 4 1 severe CF 5 2 severe CL 6 2 severe the commands that I have used for ComBat is mod = model.matrix(~as.factor(malaria), data=pheno) combat_edata = ComBat(dat=edata, batch=batch, mod=mod, numCovs=NULL, par.prior=TRUE, prior.plots=FALSE) head(mod) looks like this (Intercept) as.factor(malaria)uncomplicated AL 1 0 AO 1 0 AP 1 0 AQ 1 0 CF 1 0 CL 1 0 Why I am getting this error meassage? Please help me out. When I am taking the larger sample size (n=33) I could able to remove the batch effects without an error message but a subset of those samples giving me the above problem. I have gone through the Combat user group forum and could able to see that one or two users faced the same problem but I could not rectify it after following suggestions in those posts. Please help me out. With best regards Amit -- Amit Kumar Subudhi Research Scholar, CSIR-Senior Research Fellow, Molecular Parasitology and Systems Biology Lab, Department of Biological Sciences , FD III, BITS, Pilani, Rajasthan- 333031 e mail- amit4help@gmail.com amit.subudhi@pilani.bits-pilani.ac.in Mob No- 919983525845 -- Amit Kumar Subudhi Research Scholar, CSIR-Senior Research Fellow, Molecular Parasitology and Systems Biology Lab, Department of Biological Sciences , FD III, BITS, Pilani, Rajasthan- 333031 e mail- amit4help@gmail.com amit.subudhi@pilani.bits-pilani.ac.in Mob No- 919983525845 -- Amit Kumar Subudhi Research Scholar, CSIR-Senior Research Fellow, Molecular Parasitology and Systems Biology Lab, Department of Biological Sciences , FD III, BITS, Pilani, Rajasthan- 333031 e mail- amit4help@gmail.com amit.subudhi@pilani.bits-pilani.ac.in Mob No- 91998352584 [[alternative HTML version deleted]]