I have some single end mouse RNA sequencing data that I would like to use galaxy to analyze. I originally attempted using TopHat for alignment and was getting pretty low alignment rates (30-40%) for all of my files. After reading on this forum and other places, I saw that HISAT2 should be used. I thought that maybe this would improve my percentages since it is a more sensitive tool. However, I have run my files through and my some results improved, some are about the same, and some got worse. My overall alignment percentages range from as low as 18% to the highest being around 80%, which still seems fairly low compared to what I expected/what I have seen in others' projects. I am using the indexed reference genome (mm10). My files are fastq illumina 1.8+ scaled. Some additional notes: The alignment rates are not much different with trimming, some are even less than they were without it. I have used default parameters and read the hisat manual and changed a few parameters that I thought may help improve the alignment percentage but they did not. I attempted to BLAST some of the unaligned sequences from the samples that produced low alignment percentages and the sequences that I did BLAST either returned with no significant results or were mouse rRNA sequences, which leads me to believe that the polyA enrichment did not work as well as it could/should have. The fastq files are concatenated datasets since each sample was run in 4 lanes. I also tried aligning one sample's four files separately with HISAT2, instead of as a concatenated set, but each file's result had very low alignment percentages as well (about 4%) as opposed to only 1 having poor alignment.
I just wanted to ask if anyone had any suggestions on how to go about working with this - my sequencing core suggested demultiplexing the files and generating fastq manually with bcl2fastq (the ones I am currently using were generated on basespace), so I am working on that, but I wanted to get some other suggestions in case that is not successful.
Thank you in advance!!