Hello to everyone,
I’m using HISAT2 to analyze 3 conditions with 3 biological replicates each (A, B and C). Reads come from an Illumina paired-end analysis, trimmed with Trimmomatic.
HISAT2 works for some replicates, but not for others. WHY? Any suggestions?
Thanks a lot,
Francesco
History: Galaxy
Hi @FraCoppo
Thanks for sharing the history! Very helpful.
The error message is reporting that the input paired end reads did not have intact pairs.
Screenshot
We have a few topics about the issue. Searching with the error message can find them.
It looks like you used Trimmomatic for the QC. This tool will output intact pairs, but only if you input the reads from the same exact run. Your appear to be from mixed runs, even if they are putatively the same sample. You might be able to filter after if that was on purpose with a tool like Seqtk.
Or, you could switch to using a QA tool that will perform the clip and the filter all together. Cutadapt and fastp are two examples. Using collections can also help to avoid mixing up samples.
We have resources for this, and a simple workflow you could adapt to do everything in a batch. Change the trimming options in the Cutadapt tool to fit your data, or even swap out the trimming tool to use fastp instead if you want.
Hope this helps but let us know if you have any follow up questions! 
Hi @jennaj
thanks a lot for you suggestions!
I think that the best way is to perform a new clip + filter analysis using Cutadapt or fastp. I will update you about it.
Do you think one of them might be better for my analysis? it is RNAseq on bacteria.
Hi @FraCoppo
The Cutadapt already in the workflow I shared should be fine. Maybe start with that?
An example for the parameter tuning with bacterial data is in this tutorial. They applied a more stringent length. What you will need will depend on your FastQC results, or maybe run through the defaults and more stringent criteria (both) to see what produces the cleanest DE results later.
Glad this helped!
