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Old 04-21-2015, 01:42 PM   #1
giderk
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Default Adapting Illumina's 16s library prep protocol to function with 18s rRNA

I'm interested in studying eukaryotic parasite diversity in fecal samples that I've collected. There are a variety of Illumina myseq protocols out there, and of most interest to me is the one published by Illumina (16S metagenomic library preparation).

It seems like this protocol is cost effective and efficient, and I can't see why it would not work for 18s. My questions are...

1. For anyone that has attempted this protocol, are there particular steps to be weary of?

2. Is there any apparent reason why it would not work for 18s?

Thanks for any help.
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Old 04-22-2015, 06:41 AM   #2
microgirl123
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I've had several investigators do this with 18s. The Illumina/Nextera method works with any amplicon. You just need to be sure to pick locus primers that make an amplicon of an appropriate size. Illumina claims that you only need a 50 bp overlap - our experience with the 600-cycle kit is that the quality tanks at the ends of the reads so you want a much greater overlap.

The hardest part of the Illumina/Nextera method is getting your first round of PCR to work, especially with environmental samples. We generally recommend ordering just the locus-specific primers (without the partial adapters) first because they're cheap, optimizing your PCR with them, and then ordering the expensive tailed adapters.
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Old 04-22-2015, 09:32 AM   #3
giderk
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Thank you for responding. I just want to make sure I understand correctly. With respect to the first PCR step, you are saying that the same protocol that is optimized for just the locus-specific primers will work perfectly the same when tailed adapters are included?

Just a few follow-up questions also...

Does the second PCR reaction, adding the dual index, use the same optimized PCR protocol (but fewer cycles), or do you suggest reverting to the PCR conditions given by Illumina for adding the indices.

I would love to learn more about which universal 18s primers, and corresponding PCR conditions, your investigators have used. Are any of the papers available?

Thanks a million.
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Old 04-22-2015, 12:02 PM   #4
microgirl123
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Hopefully, the 1st step PCR will work perfectly the same with tailed primers! In reality, this is probably not the case, but if you can't get the PCR to work properly with just the locus primers, it's not going to work with the tailed primers. So we recommend testing with the cheaper locus primers to make sure it works - in our experience, once users get the locus primers to work, the tailed primers also work. I also discourage using gel extraction as a way to get around multiple bands from the first PCR - the second PCR is pretty robust, but I haven't had success using it on gel-extracted samples.

For the second round of PCR, you should use the PCR conditions outlined by Illumina. We actually use different conditions, since we "developed" the method based on a slide show from Illumina prior to them issuing the official protocol.

I've had a look online for publications from our investigators but there don't seem to be any yet - most of the runs were within the last year so it may be a bit early for papers.
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Old 04-23-2015, 08:39 AM   #5
giderk
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Thank you again. Since the work is not published yet, it would be very helpful to speak directly with you (or another person in your lab) about the 18s primers they decided to use and why. This will be my first time attempting to use this protocol (and NGS in general). If that is at all possible, I can be reached at gaet4b@umsl.edu.
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