Hi all,

I aligned my RNA-seq against reference genome using tophat, I used the default aligner bowtie2.

And also the default parameters:

    tophat -p 8 -G $annotation -o out $database L1_1.fq.gz L1_2.fq.gz

After got the results, I found out that in the unmapped.bam file, some reads have exact same sequences with the reference. The follow is one line in the unmapped.sam file:

```
DGZN8DQ1:360:H9RN8ADXX:1:1101:4791:1895 69      *       0       255     *
       *       0       0       TTTTGCTTTCTGACTCTGTGCTTGTGCCTTCAAGACTTTCACAACGATTTTCTGCTCCTCAATAAGGAAAGCCCGAGATCGGAAGAGCACACGTCTGAAC    CCCFFFFFHHHHHJJJJJJJIJJJHIJJJJJIJJJIJJJJIJJJJJIJJJJJJJJJJJJIJIJJJJJIJJJJJJHHFFDEDDDDDDDDDDDDDDDDDCCD
```

Does anyone know why the bowtie2 doesn't treat those reads as mapped? Thanks
<p>Dirty little secret: bowtie2 doesn&#39;t always find exact matches. If you change the order of reads in a file you&#39;ll sometimes get different alignment results for them. I&#39;ve never bothered to find the reason, since this ends up affecting very few reads.</p>
