Hello, I am working with RNA Seq samples from an experiment similar to that described in section 9.7 of the limma user guide (multi-level experiments). The difference is that we have multiple fastq files per subject per condition per tissue. For example:

File01a - Subject1 - diseased - tissueA File01b - Subject1 - diseased - tissueA File02a - Subject1 - diseased - tissueB File02b - Subject1 - diseased - tissueB ... Is there a recommendation on how best to analyze this set to compare diseased vs healthy? Can duplcateCorrelation be applied with block=subject tissue? Should one of the duplicate pairs be removed based on some QC measure? Is there some way to combine the data from the duplicate pairs of fastq files?

Thanks in advance, richard

When you align your fastq files you should just tell the aligner that you have more than one file per subject. All FASTQ files from a single subject/treatment/time combination should be aligned together and output as one file.