RNA-Seq data analysis pipeline

[preet]

Hello from US. I am going to start working on RNA seq data analysis in soybean. I will appreciate if someone can suggest me a good pipeline to hit count data from raw sequence data (Fastq files).....
Thanks

[westerman]

This might help.

http://www.biomedcentral.com/1471-2105/14/91

A comparison of methods for differential expression analysis of RNA-seq data
Charlotte Soneson1* and Mauro Delorenzi1,2

[GenoMax]

If you are looking for a practical guidebook then: http://en.wikibooks.org/wiki/Next_Ge..._%28NGS%29/RNA or http://www.nature.com/nprot/journal/....2012.016.html

[preet]

Thanks for suggesting the references............

[iris_aurelia]

Tophat followed by HTSeq-count will also work fine. The outcome of HTSeq-count is a list of genes with the number of reads mapped to each of them

[preet]

Thanks, what about using bowtie instead of top hat as I am only concerned about hit count data for particular gene not the splice variants.
your suggestions are much appreciated
Thanks

[iris_aurelia]

With RNA-seq you expect to find your reads on the exons of a gene. So you need to deal with the different splice variants anyway. Tophat splits the read in segments to check if for example the first half of the read maps to exon A and the second half to exon B.
Bowtie does just do the mapping to the reference genome.
After the alignment you can use HTSeq-count to count the reads that are located on particular genes.

[preet]

Thanks........

[geyihe]

Hello all guys,

I just start making RNA-seq data analysis about borrelia. Now I want to find specific gene transcription between different conditions. How to value it? use reads count or RPKM? Which is better? which kind of software is simple ans easy to use? Can I use artemis to do with it?
Thank you so much!

[dpryan]

Hello all guys,

I just start making RNA-seq data analysis about borrelia. Now I want to find specific gene transcription between different conditions. How to value it? use reads count or RPKM? Which is better? which kind of software is simple ans easy to use? Can I use artemis to do with it?
Thank you so much!

DESeq (or DESeq2), edgeR, limma (look at the voom function), etc. I would recommend raw counts rather than rpkm.

[shi]

Hello from US. I am going to start working on RNA seq data analysis in soybean. I will appreciate if someone can suggest me a good pipeline to hit count data from raw sequence data (Fastq files).....
Thanks

Hi Preet, you may consider using the Rsubread+limma/edgeR pipeline. You may use the Rsubread package to align the reads (align function) and summarize mapped reads to genes (featureCounts function), and then use limma to perform differential expression analysis.

Cheers,
Wei

[obscurite]

geyihe, I'm associated with a lab that's working on Borrelia -- please get in touch if you get a chance.

[Jeremy]

If you are looking for differential expression between two or more groups, don't forget you NEED biological replicates.

[super0925]

rna-seq pipeline

Hi I am a rookie bioinformatics who will process the rna-seq from Ion-proton(Single-ended).
The pipeline for processing which I know until now is
Bowtie, Tophat, Cufflinks, and cummeRbund for RNA-seq alignment, assembly, DE analysis, and .visualization.
Any other options?
Or What is this advantage or disadvantage of this pipeline?
Thank you!

[Zapages]

I found this guide to be very useful in understanding RNA-Seq experiment design and analysis: http://rnaseq.uoregon.edu/index.html

I hope you the best.