Ziping,

This sounds an interesting project, but could be very challenge in some
aspects. First, you have an uncompleted genome; Second, your data will be
generated from more than one platforms; Third, you are tackling a problem
with 36 conditions or time points. In regarding to the third issue, I
believe that many of excellent statisticians in this group could help you
out. I just want to add some comments for discussion.

1. Reference sequence library
Without a reference library, it will be impossible to map the short reads
generated from any platform you mentioned, PacBio may be an exception if the
reads are long enough and errors are very low. The strategy to construct a
cDNA sequencing library may also be problematic because of so many
uncertainties, for example the low expressors may not be enriched to the
lever that can give you enough coverage. Will it be possible to finish the
genome sequencing for the species you are studying? Will the cost be really
effective comparing to the genome sequencing? The model you are using
doesn't seem to have a big genome (2000mb). If you think that constructing a
transcripts library is something you should do, you should consider to use a
platform which can generate the reads as long as possible.

2. platform variations
When sequencing, it may be a good idea to use same platform for all your
samples of a project. The platform variations may affect the downstream data
analysis. For mapping or sequencing discovery, it may not be a big deal. But
for counting and transcriptome profiling, platform variation will make
downstream data analysis a little bit hard.

In short, I would like to construct reference library first; second, stick
with one platform for a specific project. In terms of the sample replicates
issue, I don't think you will need technical replicates unless you have
other purposes. People usually use three biological replicates for their
microarray studies. I would say that three biological replicates might be
minimum number for the study. Statistician may have something to say for
this issue. This is just my thought and hope it helps.

Thanks,
Steve

On Fri, Jul 23, 2010 at 3:18 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
>
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