I am using galaxy to analyze RNA seq of 100bp sing end data, sequenced with Illumina 2500. I started by uploading bam files and using the tool "Convert from BAM to FastQ" to convert my data into FastQ format. Next I used "FastQ groomer" to convert my data into one format that contains Sanger-scaled quality values with ASCII. Finally I used TopHat with the default settings and the align summary is the following:
Reads: Input : 14520796 Mapped : 6298973 (43.4% of input) of these: 1080332 (17.2%) have multiple alignments (0 have >20) 43.4% overall read mapping rate.
I think the percentage of mapped sequences is very low. Could you please give me some tips on how I should alter the TopHat parameters to improve my results?
Thank you, Diana