I’m a new user using Galaxy Europe. I am working through the tutorials then applying my learning to my own data.
I have run bowtie 2 on my filtered by quality file (single end reads) but puzzled by the output. I don’t understand what the warning means and the overall alignment rate seems low.
Any advice?
Welcome @Alison
Glad you are finding the tutorials helpful! 
In your logs, these warnings can be safely ignored. They are related to the dependencies for the tool at the EU clusters. The administrators control these options so consider details about the technical part as a notification layer. Later on, if a tool fails, and you see notes like this but there isn’t anything else in the logs indicating what is going wrong, you can ask here for feedback. Sometimes there is some problem! But I don’t see one here.
For the remainder, the statistics indicate that you have single-end reads, and the mapping rate is pretty good! Some reads didn’t map, some multi-mapped, and the others, the majority, were successfully aligned. If you ran some QA on your reads already, then this could certainly be a real result and enough for downstream steps.
Hope this helps!
Thanks Jennifer. I ran fastQC and filter by quality on the data prior to running Bowtie2 and the stats looks good (4% discarded with settings of 32 and 80%). I was slightly surprised that only 77% of reads aligned. What would you normally expect?
Hi @Alison
Thank you for sharing more details!
To evaluate more, you could convert to a bigWig file and review the hits against known feature annotation in a genome browser. Then, depending on your protocol, there might be another filtering step on the BAM itself!
I’m not sure which tutorial you are following, but both of these dive a bit deeper into assessing results through filtering, mapping, then more filtering and refinement steps.
Hope this helps! 
Fastq data with more details via FastQC
More about how to inspect analysis artifacts (fastq, BAM, vcf).
Review of mapping and review in a genome browser:
Thanks @Jennaj. I should have said this is RNAseq data. I looked only at the intro tutorials and realised from a later tutorial on RNAseq that Bowtie2 is not ‘spliceaware’ so its likely the non mapped reads span introns so I will try TopHat2 or HiSAT2. I should also try adapter trimming perhaps too. There’s a lot to learn but the tutorials and help board is very useful
