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Old 01-07-2014, 06:54 AM   #1
Gian77
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Default Suggested approach for illumina MiSeq library preparation for microbial communities s

Hi all,
I am sorry if my question sould sound stupid but I am rather new to NGS technologies and illumina platform
We are going to perform a micorbial community ecology study. We have a set of 48 samples that should be selectively amplified for variable 16S DNA regions.
Could anyone have suggest a suitable and cheap solution for this kind of amplicon sequencing approach?
I would like to reduce the number of libraries by multiplexing indexed samples, but many protocols are described in literature... Which one is the best solution for you own experiences?
Thanks a lot in advance,
Gian
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Old 01-07-2014, 07:48 AM   #2
GenoMax
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You will get multiple suggestions from different folks on the forum but I will get things started with this: http://www.nature.com/nmeth/journal/...meth.2634.html

Last edited by GenoMax; 01-07-2014 at 07:52 AM.
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Old 01-07-2014, 09:42 AM   #3
microgirl123
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Illumina has a published method. We've been using an adaptation of this method for a while now and it works well. The biggest hurdle is to get the first round of PCR working.

https://my.illumina.com/MyIllumina/B...mic-sequencing
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Old 01-07-2014, 10:18 AM   #4
GenoMax
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@microgirl123 meant to probably link to this app note: http://res.illumina.com/documents/pr..._miseq_16s.pdf

If not please correct.
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Old 01-07-2014, 10:24 AM   #5
microgirl123
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@GenoMax - what I linked is the link to the actual protocol that the app note refers to.

Illumina now has a step-by-step protocol for creating 16s (or any other amplicon) libraries. The only thing you need to buy from Illumina is the Nextera adapters. This should be really helpful for labs that are just starting out on PCR amplicon sequencing.

ETA: You might need an Illumina account (free) to access the bulletin and protocol.
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Old 01-08-2014, 05:37 AM   #6
MU Core
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These articles are good references.

http://www.pnas.org/content/early/2010/06/02/1000080107

http://genome.cshlp.org/content/21/3/494.full.html

Our biggest hurdle was identifying the extraction methods that effectively removed the PCR inhibitors from the sample.
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Old 01-08-2014, 08:16 AM   #7
Gian77
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Thanks a lot for your replies. My biggest perplexity is also related to find a good and relatively cheap sequencing service that has an experience on amplicon sequencing on the illumina miseq platform. Do you have some tips on this?
Thanks again for all your replies,
Gian
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Old 01-14-2014, 07:39 PM   #8
ScottC
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MU Core... are you willing to share the methods you've found for inhibitor removal? This is our biggest hurdle with 16S survey sequencing projects, too.
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Old 01-15-2014, 08:15 AM   #9
MU Core
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Scott, our facility doesn't perform the extractions in-house but works with the researchers to develop the best method in their own lab.

We do still see researcher to researcher variability for PCR failure rates which we believe to be due to inhibitors. The vast majority of our samples are mouse fecal samples. Our most consistent group is performing a manual precipitations with ammonium acetate and isopropanol, followed by purification over a DNeasy column; a basic extraction method. I believe the success with this method has quite a bit to do the with experience level of the individual performing the extractions.

Side-by-side comparisons were performed with commercial kits. The manual extraction method did provide higher yields and cost less. If a commercial kit is the option you would like to pursue, I recommend speaking with the folks at MO BIO. There inhibitor removal technology performed well with kits covering a wide range of environmental samples.
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Old 01-15-2014, 12:46 PM   #10
ScottC
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Hi MU Core,

We don't perform the extractions either... but, like you, we do have to deal with the poor quality samples that come our way from customers, from time to time. We usually recommend the MoBio kits, but I'm always interested to hear people's experiences. Unfortuantely, our samples come from very diverse sources so I doubt there will be a single method that suits all. Often we're dealing with very low concentrations of DNA too, which complicates things.
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