[Arthropod] EST assembly from Illumina

Mark Blaxter via arthropod%40net.bio.net (by mark.blaxter from ed.ac.uk)
Wed Dec 9 14:44:14 EST 2009


Hi

I would recommend (currently) the
(a) 454 transcriptome to build reference and
(b) SOLEXA RNASeq to refine reference and count transcripts
route

The technology for transcriptome assembly with illumina SOLEXA is  
still less-than-robust, but this will change as software such as ABySS  
gets better at it, Velvet starts to cope with different levels of  
depth across sequences and, most importantly) we start to be able to  
get good 100-base reads from paired end RNASeq.

An alternative to RNASeq for counting is deepSAGE (NlaIII tags aka),  
and is often good enough for non-model oragisms where the initial  
questions are more coarse-grained than those asked of the human genome  
or model nonvertebrates. deepSAGE also requires fewer reads per sample/ 
replicate (~1 million compared to ~5 million) and so one gets more  
'bang for your buck' in sequencing. Mapping NlaIII tags to 454  
transcriptomes works well as the cDNA template for 454 is usually  
prepared using polyA and thus includes a good representation of 3'ends.

Mark

On 9 Dec 2009, at 17:49, Yannick Wurm wrote:

> Hello again,
>
> have another question.
> We're thinking of doing some gene expression analyses for a species  
> on which we have no sequence data and the closest sequenced relative  
> is 100+ million years away.
>
> I'm thinking it may be possible to do everything in one shot:
> 	1. RNAseq (using Illumina)  on our 2 conditions of interest
> 	2. Assembly of the RNAseq data to get good gene models -> annotate
> 	3. "classic" RNAseq analysis where to identify differential  
> expression
>
> The alternative would be to first perform some 454 of a normalized  
> library to get a good overview of the transcriptome.
>
> Any experience with this? Do you think it's feasible?
>
> Kind regards,
> Yannick
>
>
> --------------------------------------------
>          yannick . wurm @ unil . ch
> Ant Genomics, Ecology & Evolution @ Lausanne
>   http://www.unil.ch/dee/page28685_fr.html
>
>
>
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