I am new to RNASeq analysis. I used tophat to generate BAM files from FASTQ files but in the parameter options set "Mean Inner Distance between Mate Pairs" to 150 assuming insert size to be around 300-400 and the read lengths were 100. Eventually, I input the BAM file to "CollectInsertSizeMetrics" from Picard in Galaxy which suggested an average insert size of 195.129108 with a standard deviation of 80.053801. Do you think I need to run tophat again and if yes with what value of Mean Inner Distance between Mate Pairs?
Or use bowtie first to map the forward and reverse fastq files and then use it to find the insert size and use it for tophat.
Thank you in advance, D Das.