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Old 03-03-2020, 09:27 PM   #1
kaka87dn99
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Default The default amplification sequence 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!
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Old 03-25-2020, 10:17 AM   #2
ajthomas
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It sounds like you want to do something similar to 16S rRNA sequencing. That is a well-established protocol. I don't know what your amplicon is, but you can probably get some pretty good guidance from the 16S protocols out there. You would just need to replace the 16S primer sequences with your own.
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Old 03-26-2020, 10:58 AM   #3
cmbetts
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It depends on your amplicon length and what you're trying to learn about it.

If it's shorter than ~500bp, you can amplify it directly with PCR primers with flanking Illumina adapter sequences, but you'll need to use some of the trickery of 16S protocols to stagger the base composition so that base calling doesn't go to crap.
If your amplicon is >300bp and you don't care about assembling individual amplicons, it would be technically easier and possibly cheaper to just treat it like gDNA with either Nextera or a fragmentation/ligation TruSeq-esque protocol because you can ignore the base complexity issues.
For longer amplicons, your only option is the Nextera or TruSeq type approach described above because you can't get reads long enough to stitch across the full sequence.
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