help with FLX 454

[genbio]

Hi Everybody,

I need help with FLX454. I want to get random sequences from a plant genome where no genomic data available. I would like to use these random sequences later in SSR identification which would be later my tools in phylogenetic studies. I've just started to learn about these next-generation sequencing.

My questions could be so basic about the system and the methodology of shotgun sequencing. I thought about making shotgun sequencing in FLX, but I don't have a budget enough to sequence the whole genome. So how can I use shotgun sequencing for partial sequencing of this plants genome? Should I somehow try to specify my DNA pool for sequencing ? If so how can I make this?
Could someone help me? I urgently need understand the system so please help me , thanks a lot!

[flxlex]

Hi,

Since you know nothing about your genome, you won't be able to to some sort of reduction straight away. Normally, I would have said use mRNA/cDNA sequencing to reduce the amount of sequence, bu I guess that won't work for SSRs.

There are people out there who use AFLPs and sequence the bands, but with varying success...

Good luck,

flxlex

Good luck,

[AKroxy]

To isolate microsatellites (SSRs), I think you could make magnetic beads (dynabeads) with a particular microsat sequence attached (ATATATATATAT for instance). Chop up the DNA, and isolate the DNA ATATATATAT containing DNA (and surrounding regions) with the dynabeads. You could do that for a bunch of different microsat sequences. Then you could pool all of the isolated DNA and 454 sequence it. Might be cheaper to clone and Sanger sequence though, unless you need a lot of loci. I think this is a pretty standard approach to SSR isolation.
But if you are heading into the world of nextgen sequencing, perhaps there is a next gen approach to your phylogenetic question that would save time and money and get you way more data. Why go back to old-fashioned SSRs?