Open francicco opened 3 years ago
Hi Francesco! I'm working with "old" data too (produced in 2014), so maybe I can help.
One important thing to note is that the Phred qualities used by Illumina software changed from Phred +64 to Phred +33. For example, Illumina pipelines 1.3 and 1.5 use Phred +64, and since Illumina 1.8, it changed to Phred +33.
You might want to use quality control software, such as FastQC, to check which scores they have. I also found this post suggesting how to tell the difference by looking directly to your data: http://seqanswers.com/forums/showthread.php?t=81978
I don't know what your goal is exactly, but assuming you will align the reads to a genome before running stringtie, you will want to tell the aligner that your data is scored in Phred +64 format. In Hisat2, you only need to provide a "--phred64" flag, for example.
Since your reads are not strand-specific, you don't need to tell Hisat2 that, since its the default.
After this, you can proceed as usual with stringtie.
If you need more information, just ask!
Pedro
Hi,
I'm trying to reuse some relatively "old" illumina RNA-seq data not strand-specific. What would be the best way to process them?
Thanks a lot Francesco