EST library vs. RNA Seq

[tteoh]

Hi! I am new to this site and new to next gen sequencing too. I am a postdoc at Arkansas State University, Jonesboro, AR. I am a plant molecular biologist working mostly with plants and getting into algae.

Does anyone know if there is any reference genome for algae? I will be working on a strain of algae that has very little info in the literature and no reference genome. I am interested in identifying and cloning genes in a particular pathway. I am trying to decide whether to generate a cDNA library or to do an RNA Seq. I am very familiar with the EST library approach but do not know much about RNA Seq. Almost everyone seems to think that RNA Seq is the way to go these days but will I able to find my genes of interest without a reference genome without going through a lot of trouble. Anyone has an opinion on this.

Thanks.

[pmiguel]

Does anyone know if there is any reference genome for algae? I will be working on a strain of algae that has very little info in the literature and no reference genome.

Red or green algae?
See:

http://plants.ensembl.org/info/data/ftp/index.html

One of each there. Unlikely to share enough sequence similarity with your species to be usable by standard mapping engines.

I am interested in identifying and cloning genes in a particular pathway. I am trying to decide whether to generate a cDNA library or to do an RNA Seq. I am very familiar with the EST library approach but do not know much about RNA Seq. Almost everyone seems to think that RNA Seq is the way to go these days but will I able to find my genes of interest without a reference genome without going through a lot of trouble. Anyone has an opinion on this.

Thanks.

Since there isn't really a human medical school at our Purdue campus, the majority of samples coming in are from species with no reference genome. Seems to work okay to do de novo transcriptomes on these and then annotate them. Works for 454 and Illumina (we use paired end 100 base reads). It might even work for SOLiD 5500XL data if you use the alternate RNA fragmentation method (using heat/divalent cation rather than RNAseIII). For Illumina we then map all the reads back to the BLAST2GO annotated contigs to get a transcript level profile. For 454 we just parse the .ace file that gsAssembler produces to give read counts.

It is "first pass" analysis. But would likely suffice for your purposes. Of course you then need to go back and clone your gene interest. But that should be considerably easier know the sequence of its transcript.

--
Phillip

[tteoh]

EST vs. RNA seq

Phillips,

Thanks for the info. What kind of support is out there for de novo transcriptomes? Sorry, I am a biologist with average computer skill and the computer aspect of NGS seems daunting to me.

teoh

[pmiguel]

I think most sequencing shops will either be able to do much of the bioinformatics heavy lifting or suggest a bioinformatics shop that will. Of course you can always just wait for simpler solutions to arise.

--
Phillip

[chma1112860]

Hello

You seem to be comfortable with EST. This is much newer for me.
Could you explain me or give me some documents about how are they named and how to get the genes names corresponding to a list of EST.

Thanks in advance

Martin

[pmiguel]

Hello

You seem to be comfortable with EST. This is much newer for me.
Could you explain me or give me some documents about how are they named and how to get the genes names corresponding to a list of EST.

Thanks in advance

Martin

Martin,
There are about 100 ways to generate data that might be termed "EST".

My guess is that you need to put in about 10-40 hours of research into this topic before you are going to be able to formulate an answerable question. Wikipedia isn't a bad place to start. You could spend your first hour there.

--
Phillip