combining reads from multiple lanes for DE analysis

Question

[Pappu]

I am tying to do differential expression (DE) analysis between different conditions. Now I have same samples sequenced at multiple lanes to improve coverage. I am wondering if it would be better to combine those reads from different lanes for quantification or quantify the reads separately before DE analysis. Having more samples for DE analysis would improve the statistical power I suppose.

[GenoMax]

If the same sample is on multiple lanes in the same flowcell you can safely combine those files prior to analysis. If the same sample has run on multiple lanes across flowcells you could still combine the data but you may want to use RG to keep track of FC in case there is some batch effect (there generally should not be any, but if you want to be extra careful).

Answer 1

[ATpoint]

There is no gold standard for this. You can quantify and quality control all runs separately and then sum up the counts from the technical replicates prior to feeding them into the DE analysis to make sure to technical problems occurred during the sequencing run, which is very rare in my experience. Still, do not treat these lane replicates as independent replicates in the DE analysis, as they are simply sequencing and not biological replicates. Technically this is of course possible but it would underestimate the sample dispersion and create significance where is none.

Answer 2

[swbarnes2]

In general, running the same library on different lanes of the same kind of instrument does add not technical variation. Unless there was a problem with the instrument, you should combine the data from the same sample together, no matter how many lanes it's spread out on.