Hi All,

Just a quick question re tophat- How does tophat handles the paired-end data when both the reads (left and right) are in one file. I mapped this data (still running) using tophat without separating the paired-end reads in two different files. Will top handle this or I should separate the files?

Thanks,RT
You should separate the FASTQ reads into two files for Tophat. It will not recognize them as paired-end reads if they are together in the same file.

From the <a href="http://ccb.jhu.edu/software/tophat/manual.shtml">manual</a>:

```
##Using TopHat

Usage: `tophat [options]* <genome_index_base> <reads1_1[,...,readsN_1]> [reads1_2,...readsN_2]`

When running TopHat with paired reads it is **critical** that the `*_1` files an the `*_2` files appear in separate comma-delimited lists, and that the order of the files in the two lists is the same.
```