I am very new to R and coding in general, so forgive me if my language is not 100% correct. I have 2 control files and 2 patient files in .cov format that I wish to analyze for DMRs using BiSeq. I am confused about whether I am supposed to import all 4 files at once using readBismark, and create 1 BSraw file with all 4 datasets, or if I am supposed to import all 4 files separately. I have tried importing the files separately, but I am not sure if I am doing it correctly. I am trying to follow the vignette for BiSeq but the information it contains is super brief and doesn't really help.

This is what I have been trying so far:

setwd("~/Desktop/control_nucleated RBC_data")

rrbsc1 <- readBismark("RRBS_C1_S19_bismark_bt2.bismark.cov", colData = DataFrame(row.names = "control_1"))

If anyone could tell me if I'm on the right track, or if I should be importing everything together, I would greatly appreciate it. Thanks

You are right about the vignette, and the help page for readBismark is about as unhelpful. And I also appreciate you re-posting rather than relying on a comment posted to a years-old post. Although you could have re-posted here what you posted there.

Anyway, in the other post you tried

rrbs <- readBismark("RRBS_C1_S19_bismark_bt2.bismark.cov", "RRBS_C2_S20_bismark_bt2.bismark.cov", colData = DataFrame(row.names = c("control_1", "control_2"), group = "control"))

And you got the predictable error that you had an unused argument. This is because there are only two arguments to readBismark and you have passed three! The first argument is 'files' and the second is 'colData'. You have used a combination of positional and named arguments (where a positional argument is inferred by its position in the function call, and a named argument is directly named. So you have essentially done this:

rrbs <- readBismark(files = "RRBS_C1_S19_bismark_bt2.bismark.cov", colData = DataFrame(row.names = c("control_1", "control_2"), group = "control"), <some unnamed argument that R has no idea about> =  "RRBS_C2_S20_bismark_bt2.bismark.cov")

What is not clear in the help page, and is super not helpful in the vignette is whether or not one can read in more than one file, and if so how one might do that. You could check the function body to see how the 'files' argument is processed, and that would make things clear, but that's not something that should be expected of an end user; hence the existence of help files and stuff.

Anyway, the 'files' argument is supposed to be a character vector of file names that you wish to read in. So the correct thing to do is either

rrbs <- readBismark(files = c("RRBS_C1_S19_bismark_bt2.bismark.cov", "RRBS_C2_S20_bismark_bt2.bismark.cov"), colData = DataFrame(row.names = c("control_1", "control_2"), group = "control"))

Or you can use positional arguments

rrbs <- readBismark(c("RRBS_C1_S19_bismark_bt2.bismark.cov", "RRBS_C2_S20_bismark_bt2.bismark.cov"), DataFrame(row.names = c("control_1", "control_2"), group = "control"))

Or you could mix'n'match, so long as you use a character vector for the first argument.