On Fri, Jul 23, 2010 at 1:18 PM, ziping zhang <zhangziping@hotmail.com>wrote:

>
> Dear All,
>
> I am
> trying to use next-generation sequencing platforms to
> profile gene expression patterns during embryogenesis in our fish model
> (about 36 different stages). The genome of our model is about 2000 Mb.
> Currently only a draft of the genome with 70% coverage is available. I
> am considering using the following strategies to carry out our project:
> 1.       Use
>  SMRT of Pacific Biosciences, TMS of Helicos, SOLiD3 of ABI, Genome
> Analyzer of illumina or other RNA-Seq methods to directly sequence RNA
> isolated from embryos at each stage.
> 2.       Combine
>  RNAs from embryos at different developmental stages to form an RNA
> pool. Use DSN treatment to get the normalized cDNA, use illumina to
> sequence this normalized cDNA and get a high coverage cDNA database.
> Then use SOLiD 3 SAGE to profile gene expression patterns, which the
> tags will be mapped onto the cDNA database firstly.
>
> I need advice about which strategy is better? And how shall I set up
> biological and technique replicates?
>
> Thanks a lot for any thoughts or advice.
>
> Sincerely Yours
>
> Ziping
>
>
Hi, Ziping.  This is definitely a question that you should be discussing
with a collaborator if the answers are not clear to you.  What you are
proposing is likely to be an expensive and complicated set of experiments
that require quite a bit of forethought.  I would suggest that you
collaborate with someone using one or more of these technologies to perform
RNA-seq analysis.  Also, don't forget that microarrays are actually very
good at this type of thing, so I would not off-hand exclude array technology
for such an experiment where the number of samples could easily be over 100.

Sean

	[[alternative HTML version deleted]]

