Hello everyone,

I have a data set generated from 88 samples in a single run using all 8 flow cells. All libraries were mixed together and sequenced on all the flow cells at a time.

So Basically I received 8 fastq files corresponding to each sample. I merged all 8 files for each sample and created single fastq file per sample. 

My question is, is it necessary to look at the batch effect in this situation? if yes how shall I specify batch when I am using SVA package? If you have any other suggestion, that's also welcome.

Thank you

Amol

The flow cell does not constitute a batch effect. There is no effect to remove, and no way to remove it if there was one.

If I understand correctly, you have run the same 88 samples in each of the flow cell lanes? In that case that's an optimal experimental design and goes a long way in preventing the sort of batch effects you might be worried about. I would argue that an optimal block design is the main answer to your question (http://www.genetics.org/content/185/2/405.full).

I personally do not merge at the fastq stage but do all my quality control of fastq files independently. That way you can still obtain data on the individual lanes and you could theoretically discard lanes that failed in some way. I would also do the mapping (alignment) independently (if you still do that), as you then can get your independent mapping statistics for each lane, which can again be helpful for QC. I then tend to merge my .bam files.

I agree with Gordon though, and I have never used lanes at later stages of the analysis, and I anticipate that would be futile and incorrect.