I would like to do RNA-seq differential expression analysis on an experiment run on 8 pigs (4 cases and 4 controls, not matched). I've already done the tophat/cuffdiff approach using the pig genome. My PI also wants me to try out Trinity (without using the pig genome sequence).

I noticed that trinity can take in multiple fastq files at a time. How should I run my Trinity run(s)? Do I do 8 separate Trinity runs? Two runs for the two conditions?

Thanks
If you're looking to do a DE analysis on your Trinity results, then provide all of your fastq's at the same time. Take note if you have PE reads or SE reads. They are supplied differently to Trinity on the command line. This will create a single *de novo* assembly. If you assemble each sample separately, you'll have trouble with the RSEM step when aligning the raw reads back to the assembly to get abundance estimates. You can do individual runs, if you simply just want a *de novo* assembly for that sample. But for the DE analysis, you'll need to create a single assembly, combining all samples as inputs.

Example:

if PE: `cat` all your read 1's, and `cat` all your read 2's

    Trinity --seqType fq --JM 50G --left reads_1.fq  --right reads_2.fq --CPU 6

For the DE analysis downstream you'll be able to specify replicates, and sample names.

If all fastq's are SE there is a `--single` option to use instead of specifying read orientation.

Check out [the Trinity site](http://trinityrnaseq.sourceforge.net/#typical_options); and for full usage there is a command line parameter: `--show_full_usage`

Hope this helps.