Guide/tutorial for the analysis of RNA-seq data

[Optimistix]

Thanks a lot for the nice guide and sharing it with all of us, Matt!

[nancyelatimer]

Matt - Awesome super-polished resource for those with or without experience in NGS or RNA-seq! Please feel free to share any other resources you have created. Thank you.

[flyyuan]

Thanks Matt for this nice guide, now, I am tring to analysis some soybean rna-seq data following this article. However, I am very new to this work, could anybody give me some suggestions to solve following problems:

1. I try to use makeTranscriptDbFromBiomart to get the information of soybean in phytozome database, but it seems there many organisms in phytozome database, how can I select the G.max which I need?

2.bowtie software map the RNA-seq tag to reference gene, what is the criterion for match or does not match.

thanks in advance!

[MDY]

Hi everyone,

Sorry for the slow reply, I somehow managed to miss the replies. For those asking where to get the seven fasta files used in this guide, they are using the data used in the referenced paper, Li et al 2008 ( http://www.ncbi.nlm.nih.gov/sites/en...,f1000m,isrctn ). As far as I know, the files aren't stored on GEO, but the authors were happy to send the data when contacted by email. The 7 files are 3 treated and 4 untreated lanes of RNA-seq.

Cheers,

Matt

[diya]

Very useful document for beginners in deep-sequencing

Hi Matt,

I have been searching so much for such kind of tutorial. The tutorial is very helpful.

Thanks,

Diya

[huyvuong]

Hi Matt,
Thank very much for sharing your guide. Would you please let me know the link to download the Li Prostate cancer dataset you mentioned in the guide, i.e the 7 fa files? I couldn't find them in the publication's supporting information. Thanks

[hanifk]

I have spent a lot time to find such a tutorial
but it seems that very little material is availble
thanks for your help

[poisson200]

Dear Matt,
It is a very good place for a document like this. Someone asked me to detail how to perform RNA-seq gene diff-ex analyses on short read data; this document is an excellent example. I think it will really help a lot of people and save a lot of time (I would have done a couple of things differently but that is just personal experience/preference).

Thank you for the contribution.

Actually, a Next Generation Sequencing wiki, if it does not exist already, is a great idea.

John.

[natstreet]

The guide is really useful thanks. In it you use the data from Li et al 2008 as an example dataset. Can you point me to where I could download the fasta files you detail?

[honey]

I think it is right place and is very useful

[MDY]

UPDATE

This guide is now available in wiki form on the seqanswers wiki. http://seqanswers.com/wiki/How-to/RNASeq_analysis In addition, I would urge everyone to look at the other resources available on the wiki.
It has been nearly a year since I first wrote this guide and it is already starting to show its age. The only way this will continue to be a useful resource is if we as a community take the time to keep it up to date. Already minor things like syntax changes introduced in software updates are causing some errors to creep in. I have received many emails from people wanting to know how to fix these problems, some I have been able to answer and some others have worked out for themselves. If you are one of these people, I would strongly urge you to add the correction to the wiki (however minor it may be), so future readers can benefit. I will do my best to change things that people bring to my attention. However, I am no longer working in the field of RNA-seq analysis and so my knowledge on the topic will become less and less useful, as well as the time I am able to spend on it. I am glad this guide has been useful to so many people and hope that with your help it will continue to be a useful in the future.

Kind Regards,

Matt


Hello,

I've written a guide to the analysis of RNA-seq data, for the purpose of differential expression analysis. It currently lives on our internal wiki that can't be viewed outside of our division, although printouts have been used at workshops. It is by no means perfect and very much a work in progress, but a number of people have found it helpful, so I thought it would useful to have it somewhere more publicly accessible.

I've attached a pdf version of the guide, although really what I was hoping was that someone here could suggest somewhere where it could be publicly hosted as a wiki. This area is so multifaceted and fast-moving that the only way such a guide can remain useful is if it can be constantly extended and updated.

If anyone has any suggestions about potential hosting, they can contact me at [email protected]

Cheers

Matt

Update: I've put a few extra things on our local Wiki and seeing as people here seem to be finding this useful I thought I'd post an updated version. I'm also an author on a review paper on Differential Expression using RNA-seq which people who find the guide useful, might also find relevant...

RNA-seq Review