[Arthropod] Re: EST assembly from Illumina
(by yannick.wurm from unil.ch)
Mon Dec 14 05:08:14 EST 2009
Hi Don & Mark,
thanks very much for your extensive replies.
DeepSage on Illumina may indeed be a viable approach.
There are indeed a few ant sequencing projects underway. However I think we are involved or at least know about most of them (the ant community is small). Yet only one group is currently working on the species of interest and they haven't done much molecular at all yet.
yannick . wurm @ unil . ch
Ant Genomics, Ecology & Evolution @ Lausanne
> Assembling short read RNA-Seq to full mRNA without a reference
> genome is harder. If you have longer (72+ bp) mate-paired reads
> it become easier than with shorter, single reads. Here is some
> discussion of this
>> 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 tran
> Software assemblers for this (which I've not used)
> include Velvet, newbler, SOAP, probably others.
> Your expression analysis will depend on having large enough
> transcript assemblies to distinguish genes. And you can expect
> a large fraction of differential expression to be in species/clade-specific
> Another approach, you might ask folks on this list if anyone
> is sequencing your ant genome, and would like to
> collaborate w/ your EST/RNA-seq data.
> - Don Gilbert
> -- d.gilbert--bioinformatics--indiana-u--bloomington-in-47405
> -- gilbertd from indiana.edu--http://marmot.bio.indiana.edu/
> I would recommend (currently) the
> (a) 454 transcriptome to build reference and
> (b) SOLEXA RNASeq to refine reference and count transcripts
> 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.
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