I'm running Tophat on paired end reads which I've run through Trimmomatic, so the command is something like:
tophat R1.paired.fastq.gz,R1.unpaired.fastq.gz,R2.unpaired.fastq.gz R2.paired.fastq.gz
If I count the reads in all four of those files and compare it with the number in the first line output from "samtools flagstat accepted_hits.bam" after running Tophat, the number in the flagstat output is higher than the number of total reads. From my understanding, that first number will only count a read once and any duplicate alignments will be counted in the "Secondary" number in the flagstat output. So how is this number higher than the number of reads input?