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Old 10-28-2014, 02:49 PM   #1
jachase@ucdavis.edu
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Red face Ion torrent library prep for MiSeq

Hello, I'm very new to the NGS community and was hoping to get help. Is it possible to create a DNA library using Ions 16s metagenomics kit then performing the sequencing on a MiSeq.

I am interested in the extra coverage of the 16s unit that the Ion systems promises but only have access to a MiSeq platform.

I would happily take other universal primer suggestions that can cover a broader ranges of the 16s unit that I can add barcodes and adapters to using the Nextra protocol.

Please help!
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Old 10-29-2014, 01:18 AM   #2
nucacidhunter
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To my knowledge Ion read length is 400bp. With MiSeq you can get 2x300 paired end reads and with HiSeq V2 Rapid Chemistry 2x250. Illumin has validated a protocol: http://supportres.illumina.com/docum...15044223-b.pdf
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Old 10-29-2014, 07:47 AM   #3
jachase@ucdavis.edu
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Question

Thanks @nucacidhunter. I'm fine to use Illumina's protocol, I was just hoping that someone has validated multilocus primers that can cover the entire 16s unit (or a large portion of) without discrimination. I'm finding some older papers that suggest some primer sequences, but I'm not sure why the method I'm envisioning is not more main stream.
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Old 10-29-2014, 07:59 AM   #4
luc
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I have not worked with 16S data, but I assume the data analysis could be quite difficult for multi-organism multi-amplicon experiments. I see the appeal of having one long amplicon.

Quote:
Originally Posted by jachase@ucdavis.edu View Post
Thanks @nucacidhunter. I'm fine to use Illumina's protocol, I was just hoping that someone has validated multilocus primers that can cover the entire 16s unit (or a large portion of) without discrimination. I'm finding some older papers that suggest some primer sequences, but I'm not sure why the method I'm envisioning is not more main stream.
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Old 10-29-2014, 08:48 AM   #5
jachase@ucdavis.edu
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Red face

Hi @Luc, I agree that the multi-location applification within a bacterial community should be hard to analyze so please correct me if I am mistaken...If there is a protocol/primer design that will allow for overlap between the variable regions, can't I just align the overlapping amplified regions (like in the Ion Torrent protocol)? Please note the attached image is from the ion torrent protocol.
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Old 10-29-2014, 11:30 PM   #6
luc
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Hi jachase, you might better contact some 16S experts - I am in over my head here.
I assume you could certainly "assemble" overlapping amplicons this way - but I doubt that this would resolve the majority of ambiguities. There is very likely not enough variation to be found in the overlaps.
You suggest to sequence the entire 16S region in pieces, because you need more variants; this might require you to assemble the pieces by the very small number of variants in the overlapping regions (which are likely short and likely mostly in the conserved part).
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Old 10-30-2014, 03:10 AM   #7
nucacidhunter
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The region used for metagenomics studies is mostly depend on the material (human microbiome, environmental) and labs. These types of studies only show one view of community and depending on platform and variable region(s) one can get different views from the same material. I do not know the analysis steps in Ion software, but it seems to me that assembling small multi-locus amplicons in a mixed community to reconstruct the whole 16S region for species would be erroneous, especially with Ion high error rate. In most published work using Illumina platform, one or two variable regions have been amplified so that by overlapping paired end reads the original variable region can be reconstructed for downstream analysis. In an ideal system long DNA fragment sequencing without amplification would give more realistic view of microbial communities but with current platforms it is very expensive.
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Old 10-30-2014, 08:22 PM   #8
jachase@ucdavis.edu
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thanks for the replies @luc @nacacidhunter...the wheel are still turning.
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Old 11-03-2014, 12:20 PM   #9
kerplunk412
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Bioo Scientific recently released a 16S V1-V3 kit for Illumina platforms, which amplifies these regions as a single amplicon so you don't have to worry about assembling fragments. We also have a 16S V4 kit.

For disclosure, I am an employee of Bioo Scientific.
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Old 11-04-2014, 07:21 AM   #10
cliffbeall
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Default In case anyone's interested

We did a homebrew version of V1V3, following Illumina's protocol and using a modification of the 27F primer given here:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2293150/

Here is an excel sheet of the primers we are using:
https://osu.box.com/s/m1rj8bsvyyw3ysztklfa

V1V3 is better for our purposes than V3V4, etc.
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Old 11-04-2014, 08:29 AM   #11
jachase@ucdavis.edu
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@Kerplunk412 and @cliffbeall thank you both for this very useful information! I will keep everyone updated with my progress!!!
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Old 11-05-2014, 12:43 PM   #12
cliffbeall
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Sorry folks, my spreadsheet had an error for one of the oligos, NEX_S522. I have now corrected it in that file
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Old 01-29-2015, 05:03 AM   #13
JenBarb
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Hello jachase,
Did you end up using the 16s Metagenomics kit from Ion Torrent?

We used it and used the Ion PGM as our sequencer. I have been thinking about ways to analyze the data and was just wondering if you had used it with any success?

Thanks,
Jen
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