How to regress out age and sex using limma removeBatchEffect

Question

[aropri]

I have a protein expression data frame with a metadata data frame which includes age and sex:

nph_csf_metadata =

age sex bam tau

70 f 5 2

75 m 6 1

72 m 4 1

71 f 4 2

I want to keep the bam and tau load but remove the effects of age and sex from my protein expression data using removeBatchEffect.

I have tried this:

log_norm_prot <- log10(norm_prot) log_norm_prot <- t(log_norm_prot)

design = model.matrix(~bam + tau, nph_csf_metadata)

reg_log_norm_prot = removeBatchEffect(log_norm_prot, covariates = nph_csf_metadata$age, design = design)

This helps me regress out age but how can I regress both out at the same time using removeBatchEffect. Appreciate any help.

Answer 1

[Gordon Smyth]

You just use removeBatchEffect with batch = sex, covariates = age and design = design. As the help page ?removeBatchEffect explains, the batch argument should be a categorical factor (like sex) and the covariates argument is for numerical covariates (like age).

Since you are already familiar with the covariates argument, I wonder why you didn't do the above already?

[aropri]

Thank you for your help, I used covariates but was only able to regress out one variable. Will try the method you advised and also check covariates again.

[aropri]

thank you, this worked. Appreciate your help so much.

Answer 2

[machan]

afaik, limma::removeBatchEffect can accept two batches i.e specifying batch = (first batch) and batch2 = (second batch)

[aropri]

so would first batch be batch = nph_csf_metadata$age, and second batch2 = nph_csf_metadata$sex?

[aropri]

I tried that and it computes but I feel like its not correct because it is giving me many values of infinity. Is there a way to use the covariate to regress out age and sex at the same time?

[machan]

Do you need the corrected assay? cause the removeBatchEffect affects only the assay for the visualization hence you need to extract it from the variance-stabilized assay - otherwise you can use

1. combat seq to remove the effect of the age or sex
2. you can try having your design as: ~age+sex+[the_rest_of_your_model]
3. check if there is something like the log likelihood ratio test for you case where you will use both the null and the full model;

[aropri]

Will check that out. However, since this is proteomic expression, variance stabilized transformation is not the correct way to normalize the data. It was normalized, then log-transformed to correct the skew of the data, and then I just want to remove age and sex as variables to not have an affect on the data.