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-   -   fungal ITS profiling primers and 500bp Ion Torrent amplicon sequencing (http://seqanswers.com/forums/showthread.php?t=29555)

Gorbenzer 04-22-2013 07:42 AM

fungal ITS profiling primers and 500bp Ion Torrent amplicon sequencing
 
Hi everyone,
i'm going to try ITS profiling using the primers developed here ( http://www.plosone.org/article/info:...l.pone.0040863 ) but the amplicon is ~500bp (470bp plus bacrcode and adapter sequences).

We use an Ion Torrent sequencer and we are using 200bp kits (we'll try 400bp kits soon).

Anyone knows if it's possible to use 200bp sequencing kits to sequence the first ~200bp bases of longer amplicons?

Thanks for the help!

genseq 04-23-2013 08:30 PM

Quote:

Originally Posted by Gorbenzer (Post 102544)
Hi everyone,
i'm going to try ITS profiling using the primers developed here ( http://www.plosone.org/article/info:...l.pone.0040863 ) but the amplicon is ~500bp (470bp plus bacrcode and adapter sequences).

We use an Ion Torrent sequencer and we are using 200bp kits (we'll try 400bp kits soon).

Anyone knows if it's possible to use 200bp sequencing kits to sequence the first ~200bp bases of longer amplicons?

Thanks for the help!

500 pb DNA not suitable for 200bp kits because the chips are very sensitive to the total charge of DNA molecules immobilized on the ionospheres.

MrGuy 04-23-2013 11:13 PM

It depends on what you are trying to do. If you want to retain strandedness, then I'd suggest tagging individual molecules, fragmenting, sequence, reassemble, then get on with the analysis. I haven't tested this.

If you are just interested in the first 200 bp (assuming fusion here), fragment select for 200 bp, ligate on only the P1 adapter, then get going. I haven't tested this.

Or you could redesign the primers so they only work for 200 or 400bp chemistries. I usually go this route. The question then becomes which of these sequences do you want to miss with the new PCR design? Do you have a good bioinformatician?

The real limiter here is that the emPCR reaction limits the length of the input fragment. See if you can make a fragment that fits within the specifications of the kit you are using while still answering the question you are asking.

Gorbenzer 04-24-2013 12:08 AM

Thanks for the reply,
usually we design 200bp specific primers but this time we have to amplify the fungal ITS region that is around 400/450 bp so primers can't be designed inside the variable region.

On the Ion Torrent comunity we have been suggested to try to cut by sonication or by enzime digestion the 450bp product in 200bp fragments.
We don't have a sonicator so we will try to use the cutting enzyme provided with Ion Torrent kits; The biggest problem will be try to define the correct exposure time.

MrGuy 04-24-2013 12:24 AM

That enzyme will work fine. I don't think we'll ever buy a sonicator.


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