Hi all,

I have two libraries of RNA-seq only with polyA of same tissue (leaf and leaf), and have mapped them to the genome. Most of these reads are in 3'UTR but not spread over the whole gene body. And the size of these two libraries are in great difference, like 25,000 reads versus 1250,000 reads. About 40% and 60% of genes only have 1 read in small lib and bigger one, respectively. Most of the tags are dominated by only a few genes. I want to combine these two libs for larger one, but I think I should normalize the read count before pooling them together.

If use TPM normalization, read count in smaller library will be multiplied by 50 times, that means the 1-tag gene will become 50-tag gene, in the small lib, while maybe that gene is also 1-tag gene in bigger lib, I feel not comfortable that TPM may make skew the read distribution. Do you have any idea on normalizing the data instead of TPM? 

Thanks a lot in advance. 

Regards, 
Xiaohui

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