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imendez 03-10-2016 02:46 AM

One amplicon sequencing in Illumina
 
Dear all!

I want to sequence only one amplicon using Illumina platforms (I know that the deep is going to be crazy). I have very little experience on using this kind of platforms. Does anyone have experience on doing this? Does anyone know with protocol should I follow? Any hints and advice would be appreciated.

Thanks!

Bukowski 03-11-2016 12:16 PM

Why did you post this in the Ion Torrent section then?

Why do you need an NGS approach for this?

Low diversity libraries (and this is the lowest possible diversity) are not the greatest fun to work with on Illumina platforms.

NextGenSeq 03-14-2016 05:48 AM

If he's trying to detect extremely low amounts of a mutant it may make sense.

I think the only way to do this is to amplify the region of interest with several sets of primers which give varying lengths of amplicons. It's not a single amplicon then though.

thermophile 03-14-2016 06:40 AM

Single amplicon-as in 16S? Regardless, the primer design template from this paper should work well for any target gene (check for hairpins, check seq temp, etc). Plan on running a lot of phiX and underclustering

http://aem.asm.org/content/79/17/511...0-b631ab573369

lxkx 03-22-2016 12:51 PM

If you are doing one amplicon and not plan to do it again. It is probably easier to find someone around and spike in your sample with their run.

The 16s protocol is a good reference if you want to build your own design, at least that is where I started. Low diversity is not an issue at all on Miseq. I have been doing 2-3 amplicons for my project frequently.

jtotheulie 05-27-2016 05:55 AM

If I have 16s, 18s, and its amplicons from 170 different samples and I want to do deep sequencing on MiSeq, what would be the best way to go about this?

I understand that MiSeq has an easier time sequencing low diversity libraries with V3 kit.

I want this to be cheap as possible.

How should I barcode and lib prep the samples.

How should I barcode and lip prep samples if I find they are at a low quant.


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