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Old 03-04-2013, 07:47 AM   #1
lorendarith
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Default Direct 16S amplicon sequencing?

Has anyone ever tried doing an amplicon sequencing on "non-enriched" amplicons? The idea is to basically just fragment everything, add Truseq adapters (with indexes) and then sequence using a custom sequencing primer and index 1 primer?

I do realize that getting such samples to satisfying cluster densities (of sequenceable fragments) might be very hard due to the presence of undesired fragments which would also form clusters. But what if you could reduce the overall noise of undesired fragments (but not eliminate them completely), would this direct sequencing then have more sense?

I would just like to get completely rid of the PCR bias before sequencing...

Any thoughts?
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Old 03-04-2013, 08:36 AM   #2
TonyBrooks
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Quote:
Originally Posted by lorendarith View Post
Has anyone ever tried doing an amplicon sequencing on "non-enriched" amplicons? The idea is to basically just fragment everything, add Truseq adapters (with indexes) and then sequence using a custom sequencing primer and index 1 primer?

I do realize that getting such samples to satisfying cluster densities (of sequenceable fragments) might be very hard due to the presence of undesired fragments which would also form clusters. But what if you could reduce the overall noise of undesired fragments (but not eliminate them completely), would this direct sequencing then have more sense?

I would just like to get completely rid of the PCR bias before sequencing...

Any thoughts?
So you're talking about whole genome metagenomics?
We've done that on 454, but we still use some amplification for Illumina. We thought about trying the new Illumina PCR-free kit, but the 2g input for 550bp insert is a killer.
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Old 03-04-2013, 10:41 AM   #3
lorendarith
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Originally Posted by TonyBrooks View Post
So you're talking about whole genome metagenomics?
We've done that on 454, but we still use some amplification for Illumina. We thought about trying the new Illumina PCR-free kit, but the 2g input for 550bp insert is a killer.
Yeah, it's sort of like WGS, but you wouldn't get the rest which is not 16S.
The advantage from just doing WGS would be that you have fragments on the flowcell where the 16S part starts beyond 100 or 250 bp of the adapter, you would still get this information.
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