Detecting bacterial TSS from RNA-Seq data, how?

Question

[R.Blues]

Hello everyone,

Sorry for bothering you, but I am not sure if there is a standard protocol for inferring or detecting transcriptional start sites (TSS) from bacterial RNA-Seq paired-end data, and I would be very grateful if someone could help me here. I have been searching and I am not really sure what to do! :)

The data is already mapped vs the reference genome using the gtf annotation file. I just want to determinate the +1 for the different transcripts! Is that as easy as taking the first nucleotide of each operon? Or is there a better way?

I am quite lost, and I would really appreciate any help. Thank you very much.

Answer 1

[Asaf]

There are methods to enrich for primary transcripts by catching the ppp at the 5' end of the molecule (simply digesting 5'p and 5'OH). Take a look at: http://nar.oxfordjournals.org/content/44/W1/W46.long

[R.Blues]

Yes, it's true! Thanks! However, I have to work with this RNA-Seq data, so I cannot redo the experimento. I will take this into account for the next time (thank you!) :)

Answer 2

[GouthamAtla]

You can not detect TSS from RNA-Seq data. You need data from protocols like CAGE-Seq.

In RNA-seq, the mRNA is randomly fragmented and sequenced. so the precise TSS can not be determined as the mRNA is sequenced randomly. In protocols like CAGE, they explicitly sequence the 5' end of the transcript, so we know the exact TSS with 1bp resolution and even we can quantify the dominant TSS.

[R.Blues]

I have found alternatives as TSSpredator (http://it.inf.uni-tuebingen.de/?page_id=190). I understand that it will not work as well as a CAGE, but I have to work with this RNA-Seq data.

[h.botond]

I am very interested in your experiences with the TSS predator. Is was usefule for the TSS detection with RNA-Seq data? How did you convert the bam file to .gf file format? The .gf file format is a Wiggle Traack Farmat (.wig)? Thank you for your reply!

[Anima Mundi]

Hello, this looks to me like a strong statement, could you please substantiate it?

[GouthamAtla]

I updated the answer

[Anima Mundi]

Thanks. I would also say that even if optimized protocols are by definition better at finding TSSs, TSS information can still be extracted by conventional RNA-Seq pipelines.

[GouthamAtla]

Yes. Approximate TSSs can be estimated from RNA-Seq but not precise TSS at 1 bp resolution can not be estimated. It depends on the use case whether we need precise TSS or approximate.