Hisat2 vs Tophat2 variation between tech reps

[kuristy]

I have a question regarding the variability of FPKM I am seeing between technical replicates in the Hisat2, stringtie pipeline. When I plot technical replicates by log2 FPKM, I am getting Spearman correlations of 0.6. I used the same fastq files in the tophat, cufflinks pipeline (default settings) and had Spearman correlations of 0.9. I am wondering which parameters I can change in Hisat2 to make it behave more like tophat2. In hisat, I have tried several different parameters and I am getting similar results regardless of the parameters I use.

The parameters I have tried in hisat2 are as follows:

-k 2 --max-seeds 15 --no-mixed

-k 3 --max-seeds 10

-k 10 --max-seeds 10

default settings


I am getting a high percentage of multi-mapping (~20%) and I am wondering if that is a problem.

Thank you for your help!

[kuristy]

I have answered my own question, but I am posting just in case someone else has a similar issue.

I ran the same tophat data with stringtie and got a similarly bad correlation between technical replicates of 0.62. Now I know that the problem must be with stringtie and not with hisat2.