Hi,
I just got my first NovaSeq-produced RNASeq dataset and the obtained lib sizes are quite uneven. The attached file shows the lib sizes. If you look at it, you’ll see that there are three libs between 75 and 100 M reads, another one that goes beyond the 100 M mark and, “best” of it, a library that shows ~400M reads (!!). On the lower side, one library barely gets 20M reads. The second plot in the pdf shows the number of mapped reads, reflecting the overall lib size tendencies.
The Sequencing Facility is not able to produce any plausible explanation to that 400 vs 20 M lib size range, provided that the pool behaved correctly in the MiSeq checking run. Have you ever encountered this problem? Is there any known tagwise bias in the NovaSeq?
As far as the analysis of the data is concerned, I know edgeR and DESeq2 packages deal with lib size differences but is not this too much?
I’d be grateful if you could help me out on this.
Best,
David
I just got my first NovaSeq-produced RNASeq dataset and the obtained lib sizes are quite uneven. The attached file shows the lib sizes. If you look at it, you’ll see that there are three libs between 75 and 100 M reads, another one that goes beyond the 100 M mark and, “best” of it, a library that shows ~400M reads (!!). On the lower side, one library barely gets 20M reads. The second plot in the pdf shows the number of mapped reads, reflecting the overall lib size tendencies.
The Sequencing Facility is not able to produce any plausible explanation to that 400 vs 20 M lib size range, provided that the pool behaved correctly in the MiSeq checking run. Have you ever encountered this problem? Is there any known tagwise bias in the NovaSeq?
As far as the analysis of the data is concerned, I know edgeR and DESeq2 packages deal with lib size differences but is not this too much?
I’d be grateful if you could help me out on this.
Best,
David
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