Dear All

I need some (lots) suggestions and help,  first and most important is that i am working on bacterial RNA seq (illumina reads)
my analysis steps are as following ....

Step 1.  FASTQ sequence data was groomed

Step 2. I did mapping by Bowtie with default parameters. Reference genome fasta file i am using from my history, because the reference genome is not vaialble on galaxy.

Step3. i sorted the bowtie output file using r workflow (Germy Goecks workflow)  , link below
https://main.g2.bx.psu.edu/u/jeremy/p/transcriptome-analysis-faq#faq2

Step4. this sorting provided me Concatenate files

Step5. Concatenated files were used to run CUFFLINK, this provided me assembled trancript file

Step6: Assembled transcript files from step 5 were used for CUFFMERGE

Step 7:  For CUFFDIFF Transcript GTF file generated from  Step 6 and  concatenate files from step 4 were used 

Now my question is if this workflow is acceptable for bacterial transcriptome anaylsis,

Should i filter SAM file, if yes then at which step

Should i convert SAM file to the BAM file, then at which step it should be

Is that Ok to use fasta of reference genome for mapping should it be converted to any other format, if yes then what should be the workflow

If any one have experince of bowtie parametes to map bacterial RNA seqquence analsis are very much welcomed 

Thanking you all