Prokaryotic Differential expression analysis- RNA seq data and software

Question

[zh.khodadadi]

Hi every body I want to analyze(Differential expression ) my rna seq data of bacteria single end read,50 bp, Illumina HiSeq 2000. Can I use tuxedo software? What is the pipeline of Differential expression analysis for bacteria rna seq data? thanks a lot

[GenoMax]

Since you don't need to account for splicing you could use any NGS aligner and count the reads that are aligning to the genes using an annotation file (GTF/GFF). This can then be followed by one of the popular DE analysis programs DESeq2/edgeR etc.

An online pipeline is mentioned in this thread (if that is of interest): Bacterial RNA-seq analysis

[Farbod]

Dear genomax2, Hi

Is this pipeline helpful for bacteria?

FastQC --> Trimmomatic/Cutadapt --> STAR --> Samtools --> featureCounts/HTseq-count --> DESeq2

[WouterDeCoster]

STAR can output sorted bam, so I'm not sure why you add samtools in there ;)

[Farbod]

Hi,

I have used it from other Biostars posts, so it should be :

FastQC --> Trimmomatic/Cutadapt --> STAR --> featureCounts/HTseq-count --> DESeq2

[GenoMax]

I am partial to BBMap suite so I would do

FastQC --> bbduk (from BBMap) --> bbmap (from BBMap)  --> featureCounts --> DESeq2

BBMap will output bam files directly (as long as samtools is available in $PATH, but you would need to sort them). featureCounts will sort BAM's automatically, in case you do not want to use samtools.

[WouterDeCoster]

Seems valid, but as @genomax2 already wrote a spliced aligner isn't really necessary (because no splicing in bacteria), and I also would run fastQC after trimming to see how successful it was.

[Farbod]

Dear zh.khodadadi, Hi

Do you have any reference genome for your bacteria of interest?

~ Best

[zh.khodadadi]

thanks all I am new in NGS why I dont use Tuxedo pipeline?

[zh.khodadadi]

My bacteria has reference genome and I study some posts like that genomax2 says.

[WouterDeCoster]

Please use ADD REPLY to answer to earlier comments, as such this thread remains logically structured and easy to follow.

[zh.khodadadi]

ok thanks for this guidance