First, I am brand new to this forum and brand new to RNAseq; I searched the forums for this, but didn't find another question similar enough to answer it.

I have 2 control files and 2 treatment files (RNA sequencing). The files are old enough that they unstranded files and they are not paired end files (hence each 4 are distinct).

I trimmed the files with trimmomatic, and was going to perform alignment with TopHat2 next. Our cluster has all the software installed for Bowtie2, samtools etc...

I downloaded and unzipped UCSC hg18 bowtie indexes here: http://bowtie-bio.sourceforge.net/bowtie2/manual.shtml

So, the questions.

1. Do I need to run all the files through one at a time?
2. If I run them through TH2 separately, do I have to specify 4 different output folders for each submission?

3. I ran into the thread issue with TH2 last night. I specified p=8 threads, and the submission crapped out 1 hour in

   Searching for junctions via segment mapping
   [FAILED]   Error: segment-based junction search failed with err =1
   Error: could not get read# 9850246 from stream!)
   

   I then specified p=1 and it ran, but took 6 hours....if someone knows a good sbatch parameter list to prevent this, I would greatly appreciate it.

4. Lastly, I got one warning

   Checking for reference FASTA file
   Warning: Could not find FASTA file /locationofbowtieindexes/hg18.fa)
   

   Do I need to put a genome.fa file there from here? (http://support.illumina.com/sequencing/sequencing_software/igenome.html hg38 link under Homo Sapien)?

This is my current submission script:

    #!/bin/sh
    SAMPLE_ID=trim_Mitchell_P2D-F2.fastq
    GENE_REF=filepathtobowtie2index
    P=1 #USE 8 THREADS

    tophat2 -o tophat_out -p $P $GENE_REF pathtoRNAsequencefastafile/$SAMPLE_ID

Thanks so much for your help and sorry for my noobness!