RNA-Seq: Recent advances in RNA sequence analysis.

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Recent advances in RNA sequence analysis.

F1000 Biol Rep. 2010;2:64

Authors: Salzberg SL

The latest high-throughput DNA sequencing technology can now be applied on a large scale to capture the complete set of mRNA transcripts in a cell, using a technique called RNA-seq. Although RNA-seq is only 2 years old, it has rapidly swept through the field of genomics, and it is now being used to analyze the transcriptomes of organisms ranging from bacteria to primates. The depth of sequencing allows researchers to quantify the level of expression of genes, to discover alternative isoforms in eukaryotic species, and even to characterize the operon structure of bacterial genomes.

PMID: 21173855 [PubMed - in process]



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[Rodri]

Using this introduction of recent advances in RNA sequence analysis, and specifically about the bacterial genome structure obtained with RNA-Seq analysis, my question is:

Do you Know the programs used to the bioinformatic characterization of the bacterial operon structure?

As the first answer I include the Cufflinks, that I used with the next options:

1) -g GTF-file tells cufflinks to use the reference annotations GTF file to guide the RABT assembly (Reference Annotation Bassed Transcript Assembly) "Roberts et al., 2011".
2) -I 49. The maximum intron length is 49 and the minimum intron size as default is 50 pb. Then, with this option Cufflinks don't find any intron region, not present in prokaryotic genome.

After the Cufflinks application with the 7 mapping files (bam files) obtained with TopHat using several lanes, I used Cuffmerge application taking the assembly GTF files obtained with Cufflinks as input, and I obtained a merged GTF file that includes the operon distribution.

I like to compare the results with other possibilities and in the next reference (Passalaqua et al., 2009), we find in "The Quality control and statistical and bioinformatic analysis of sequence data" (Page 3204) that "the statistical analyses were done using R, Microsoft Excell 2008, and GraphPad Prism 5.0a. Processsing of sequence data for mapping or quantification was done using custom PERL scripts that are avaible for download at http://bergmanlab.biology.gatech.edu". This page is out of service and after mailing the correspondig author nickbergman, I don't receive any answer. If you have the scripts used and referenced, I really grate to receive them.

Thanks for your aid.

[nivea]

Hey,

I don't think using Cufflinks reference based method to reconstruct operon is a good idea, since if you use cuffmerge the output should be the "merged" operon structures from all the conditions or time points, then how can you determine the operon dynamic structure?Maybe Cufflinks without a reference genome could be an option for bacterial transcriptome construction.

But I do have question here, does anyone try Scripture for the operon construction in bacteria?

Using this introduction of recent advances in RNA sequence analysis, and specifically about the bacterial genome structure obtained with RNA-Seq analysis, my question is:

Do you Know the programs used to the bioinformatic characterization of the bacterial operon structure?

As the first answer I include the Cufflinks, that I used with the next options:

1) -g GTF-file tells cufflinks to use the reference annotations GTF file to guide the RABT assembly (Reference Annotation Bassed Transcript Assembly) "Roberts et al., 2011".
2) -I 49. The maximum intron length is 49 and the minimum intron size as default is 50 pb. Then, with this option Cufflinks don't find any intron region, not present in prokaryotic genome.

After the Cufflinks application with the 7 mapping files (bam files) obtained with TopHat using several lanes, I used Cuffmerge application taking the assembly GTF files obtained with Cufflinks as input, and I obtained a merged GTF file that includes the operon distribution.

I like to compare the results with other possibilities and in the next reference (Passalaqua et al., 2009), we find in "The Quality control and statistical and bioinformatic analysis of sequence data" (Page 3204) that "the statistical analyses were done using R, Microsoft Excell 2008, and GraphPad Prism 5.0a. Processsing of sequence data for mapping or quantification was done using custom PERL scripts that are avaible for download at http://bergmanlab.biology.gatech.edu". This page is out of service and after mailing the correspondig author nickbergman, I don't receive any answer. If you have the scripts used and referenced, I really grate to receive them.

Thanks for your aid.