Input Paired-ended data to Tophat

[annavilborg]

Hi,

This should really be an easy thing, but I am getting conflicting advice...so my question is: What is the correct way to input paired-ended (75bp, Illumina Hi-Seq) strand specific data to Tophat? Should I enter the separate file lists for R1 and R2 fastq files? Or should I use a merged file (that is also available for download from our sequencing facility) and input the same file twice?

The first option seems more logical to me, but I have heard and read that if one inputs the same file twice, Tophat should be able to figure out that this is paired-ended data, split it (I assume) and use that.

Does anyone know the correct approach?

Thank you!

[mknut]

From the manual:

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

<reads1_1[,...,readsN_1]> A comma-separated list of files containing reads in FASTQ or FASTA format. When running TopHat with paired-end reads, this should be the *_1 ("left") set of files.
<[reads1_2,...readsN_2]> A comma-separated list of files containing reads in FASTA or FASTA format. Only used when running TopHat with paired end reads, and contains the *_2 ("right") set of files. The *_2 files MUST appear in the same order as the *_1 files.

So for example:

tophat -o outputDirectoryName -r 50 --library-type fr-unstranded /path/to/genome/reference /path/to/first/read/read_1.fastq /path/to/second/read/read_2.fastq

Having a quick look at the tophat sourcecode (prep_reads.cpp), it looks like tophat processes reads sequentially, ie. the first read in read_1.fastq has to be the mate of the first read in read_2.fastq. Tophat does not seem to be doing any automatic read mate matching, at least nothing I can see. I think that if you merge the reads into one file and submit it twice as read1 and read2, you will lose synchronisation between the reads, if it works at all. Anyway, the method above is the method suggested in the manual, so I would stick to it.
To reiterate:

Should I enter the separate file lists for R1 and R2 fastq files?

Yes

[annavilborg]

Thank you! And thanks for explaining how tophat does this, great to get the background. I'll go ahead and run my samples as separate R1 and R2 files.