How is the normalization and expression calculation in the Rockhopper ?

Question

[brendarc]

Hey everyone, I'm using Rockhopper to do my RNAseq analysis, with 3 conditions and biological triplicates. I'm quite in doubt of how is made the normalization and expression calculation? For the final expression of each gene, looks that they use the total of the reads of the biological triplicate to calculate the expression, wouldnt be average? Thanks, Brenda

[h.mon]

For the final expression of each gene, looks that they use the total of the reads of the biological triplicate to calculate the expression, wouldnt be average?

How did you reach this conclusion? Could you explain?

[brendarc]

Hey, thanks for replying to me! Actually, I´m trying to figure out how to get the right values, but I got only close values. I only got more close values, when I´m using the sum of the reads for the three triplicates, to calculate RPKM, and then at the end, I´m doing the average, but the values are just close. For example, i have data for one gene, in biological triplicate: Raw counts (278 ; 35; 1276), Normalization values (they said they calculate using upper quartile, but I couldn´t get using raw counts/upper quartile) (11621; 1664 ; 53370); and RPKM (7), Expression value (13). The total reads from triplicates are ( 6948210; 5455341; 8405162), upper quartile from each (1286; 973.5; 1927). And the gene length is 1600 bp. Then, finally, I definitely didn't get the same normalization values, either expression values, and only RPKM calculation seems reasonable. If you could help me with any idea, it would be really helpful! Best, Brenda

Answer 1

[h.mon]

Did you read the User Guide, FAQ and papers? For example, from the FAQ:

What do the expression values for each transcript correspond to?

Expression values reported by Rockhopper for each transcript in each condition are similar to RPKM (reads per kilobase per million mapped reads) values. However, RPKM values are generally normalized by the total mapped reads, whereas the expression values reported by Rockhopper are normalized by the upper quartile of gene expression, which is a more robust normalizer.