Normalizing Illumina RNA libraries

[sinnafoch]

Hi,

I am relatively new to the Genomics/Transcriptomics game and about to prep Illumina TrueSeq Total RNA libraries from fungal RNA. I read through the instructions several times, everything seems quite clear to me except the Normalize/Pooling step at the very end.

Isn't the normalization step just important when doing comparative Transcriptomics?
I need transcriptomes just to assist genome annotation, so normalization is not a big issue. Am I right?


Thank you for your help!

[JeremyDay]

The final normalization is for pooling multiplexed samples. Its main purpose is to pool each individual library at equimolar concentrations. Illumina normally advises normalizing the libraries to the same concentration, then pooling by combining same volumes of each library. Or, you can quantitate each library and combine different volumes after calculating, however, do not do this method if your library concentrations have a high range. You could potentially pool without doing this, but then you run the risk of some libraries having lots of reads and displacing the reads required for other libraries. If some libraries require more reads or less, you can account for this when pooling.

[woshiliangliang]

Hi sinnafoch,
i think you mistake the 2 application of the word "normalization".
as you metioned in your question,
if you need transcriptomes to assist genome annotation,
you also can perform a normalization step in order to reduce the risk of sequencing only highly expressed genes.
meanwhile the results cannot show actual gene expression levels between samples.