Regulatory RNA prediction steps in bacterial genome sequences

Question

[rahimitouraj]

Dear I work on Bacillus subtilis genome sequences, and want to use a pipeline for prediction small Regulatory Rna and relation with Transcription factor in intergenic region,Are there any pipeline for prediction and analysis relation of these regulatory elemnets?

Answer 1

[Asaf]

You have the 2001 approach: Predict rho-independent terminators after IGRs with a nice fold (Argaman et al., 2001; Rivas et al., 2001; Wassarman et al., 2001).

Nowdays you can use RNAseq results (you can find plenty of those for B. subtilis in different conditions) and generate a transcriptome gff file and subtract the gff file of the coding regions from the gff you got to get a list of sRNAs. You can use RNApol ChIP-seq to get orphan transcription start site to reinforce your findings (B. Palsson did such things in E. coli, e.g. http://www.ncbi.nlm.nih.gov/pubmed/22912590)

[rahimitouraj]

Thanks for your reply Asaf, Is these method is a right way for do it: download small RNA for B.subtilis starin (168 or other ) from BSRD http://www.bac-srna.org/BSRD/index.jsp# but this site is down now, other source Rfam(also down for maintenance) then I searched this sequences(small RNA) in our bacterial genome(as BLAST), After that extract orthologus genes and run clastal? for multiple sequence alignment. After that use infernal for prediction putative RNA. Best

[Asaf]

You can run cmscan on the genome to search for known sRNA profile, should work better than BLAST but then you won't find novel ones. Looking at RNAseq results should work if you are looking at a wide range of conditions, their expression is usually very high in the right condition.

[rahimitouraj]

Thanks , I'm a beginner in these era , How I can generate transcriptome gff from RNAseq data.

[rahimitouraj]

Dear I downloaded SRA data for bacillus subtilis from NCBI SRA and convert it to fastq file then mapped by clc bio to our bacterial genome then I create track list file and extract region with mapped IGR , is these correct method? Best

[Asaf]

Sounds good, did you find something? Have a look at a genome browser to validate your hits.

[rahimitouraj]

Hi, Thanks Yes I saw some RNA in IGR in our genome and reference genome, But now i want to find a solution for extact these region from CLC bio and compare between two species(our genome and ref. genome). then try to validate them. Best