Rna-Seq Analysis With Bacteria, Rrna Reduced, Strand Specific

Question

[epistatic]

I do mostly eukaryotic RNA-seq using Tophat2 and then cufflinks, DEseq, edgeR, etc.

I have some bacterial rRNA- (Ribo-zero) and TruSeq directional (strand-specific) samples, PE-50 on my HiSeq 2500. I am not sure the best way to go about the alignment since these have no introns, do I use bowtie2 or bwa directly or should I still use a GTF and Tophat2?

For GTF files, is there a standard location to retrieve these? If I have a GFF can I use it or do I need to format into a GTF?

Thank you!

Answer 1

[epistatic]

EDGE-pro, Estimated Degree of Gene Expression in PROkaryots -> DESeq works great for Bacterial RNA-seq. It uses bowtie for the alignment and includes a script to take the FPKM output and create a count table that you can immediately upload into R. I also had a bacterial genome with two plasmids and it handled it all extremely well.

Input is genome fasta, ptt, rnt, and read fastq

http://ccb.jhu.edu/software/EDGE-pro/

Answer 2

[Mathew Bunj]

Bowtie 2 should be fine, I dont think you need TopHat2, BTW what was the purpose of Ribo zero library?

[Torst]

The RiboZero library will remove the ribosomal RNA, which typically makes up >95% of the total RNA. Unlike with eukaryotes, we can't enrich for mRNA using polyA tails, so we deplete rRNA instead.

Answer 3

[epistatic]

Response to different anti-microbial agents.