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Old 08-14-2013, 12:10 AM   #1
Catherine_Burke
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Default Polymerase of choice for generating 16S amplicons?

I'm currently generating 16S amplicon libraries for Illumin sequencing using Phusion polymerase, but we have noticed a high level of recombination during the enrichment PCR (~30% of sequences have the incorrect barcode combinations).

I've since read that proof-reading polymerases can give a higher chance of chimera formation. From the literature it doesn't seem like anyone is particularly using any of the published recommendations for avoiding chimera formation (longer extension times, low template input and limited PCR cycles).

I'm wondering if there is a particular polymerase that people prefer for generating 16S amplicons which gives both high fidelity and low chimera formation?

Thanks,

Cath
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Old 08-14-2013, 03:33 AM   #2
addyblanch
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Could look at KAPA HI-FI?

http://www.kapabiosystems.com/produc...-hifi-pcr-kits

They have become NGS library prep gold standard very rapidly because of their low error rate and minimal GC bias.

Have a look at the Mike Quail paper below:

http://www.nature.com/nmeth/journal/...meth.1814.html
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Old 08-14-2013, 07:08 AM   #3
bstamps
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Honestly? Fermentas Taq. Nothing special- when you think about it you're generating a 300-500bp fragment, and the likelihood of a misincorporation is low enough that I can live with it. It works well and our libraries usually look very good. Interesting about phusion and the issue of barcode swapping- any specific reference for the higher rate of chimera formation?
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Old 08-14-2013, 10:24 AM   #4
ATϟGC
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Here is an article, in case you do not have it already, by Lahr and Katz (2009) that indicates using fewer cycles and lower template addition minimizes or eliminates chimera formation:

Biotechniques. 2009 Oct;47(4):857-66. doi: 10.2144/000113219.
Reducing the impact of PCR-mediated recombination in molecular evolution and environmental studies using a new-generation high-fidelity DNA polymerase.

(http://www.biotechniques.com/Biotech...es-176950.html)

Lahr and Katz used extension times 3x longer than recommended by the Finnzymes but did not vary extension times to investigate its effects. However, I seem to remember reading a few years ago in a Phusion High Fidelity product brochure that you should not exceed the recommended extension times as more errors may be incorporated.

I have no experience myself with minimization of chimeric amplicons but I have found Phusion hot start to be particularly effective at minimizing stutter in sanger sequencing past homopolymer runs (<17 bp) and to be generally robust to inhibitors. I do, however, find it to be a bit picky with what primer-pairs it will work well with (Perhaps due to mis-matching and resulting endonuclease activity at the site of mis-match?)

Here is a ref for the use of Phusion and Herculase for minimizing homopolymer stuttering:
http://www.biotechniques.com/Biotech...es-197505.html
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Old 08-14-2013, 03:33 PM   #5
Catherine_Burke
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Thanks for all your replies.

I did see the Lahr and Katz (2009) paper, which is what got me worried about phusion! They do show very little recombination with small amounts of starting template, but I'm unlikely to have less than 10^5 copies in an enrichment PCR. Above this template concentration, they see ~50% chimera formation with 39 cycles (they didn't test less than this).

Another paper indicates that recombination will be more of a problem with more complex templates (like microbial communities!)
http://www.ncbi.nlm.nih.gov/pubmed/22278883

And here is the link to the paper which suggests proofreading polymerases cause more chimeras than regular taq (although I don't have access so have only read the abstract!)
http://link.springer.com/article/10....275-012-2642-z

From speaking to a few other people, it seems like most use a regular old Taq, so I'll probably try that for next time...
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Old 08-14-2013, 03:34 PM   #6
Catherine_Burke
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"Above this template concentration, they see ~50% chimera formation with 39 cycles (they didn't test less than this). "

Sorry, that should have read 30 cycles...
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Old 08-15-2013, 03:37 AM   #7
JamieHeather
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Another factor to consider is the ramp speed; slower temperature might lead to fewer chimeras.


http://www.sciencedirect.com/science/article/pii/S0167701213001073
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Old 03-14-2014, 11:34 AM   #8
replicate
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From the Lahr paper above, the indication is that chimeras are a pitfall of PCR generally, and while the paper uses next-gen polymerases, this is simply because no-one has(had) looked at recombination and Phu etc. before - is there anything suggesting that hi-fidelity polymerases are more prone to chimera-formation/dropping extension runs?
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Old 03-17-2014, 05:10 PM   #9
Catherine_Burke
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"is there anything suggesting that hi-fidelity polymerases are more prone to chimera-formation/dropping extension runs?"

The link given above (here again http://link.springer.com/article/10....275-012-2642-z) does suggest that proof reading polymerases have a higher rate of recombination. Also from our own data, we saw much less recombination with regular Taq (we used Qiagen) than with Phusion. This was based on the % reads which had incorrect barcode combinations (i.e. recombined in the pooled enrichment PCR).
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Old 03-04-2016, 08:09 PM   #10
peterwang
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Bumping this old thread, which I came across in reading about artifactual PCR recombination that may occur in generating amplicon libraries.

I will add another paper that was helpful for framing my thinking about this:

Smyth RP, Schlub TE, Grimm A, Venturi V, Chopra A, Mallal S, Davenport MP, Mak J.
Reducing chimera formation during PCR amplification to ensure accurate genotyping.
Gene. 2010 Dec 1;469(1-2):45-51. doi: 10.1016/j.gene.2010.08.009.
http://www.sciencedirect.com/science...78111910003616

They assert that "[chimeras] generally form during the latter plateau stages of PCR when dNTPs and primer are exhausted and PCR inhibitors have accumulated." Their solution was to carefully assess the point after which the PCR ceases to be exponential; in their case, this was 29 cycles for an input of 2500 copies. Raising the input 5-fold (equivalent to doing 2.3 extra cycles) caused a 7.5-fold increase in the recombination frequency.

Their PCRs used Phusion polymerase, high concentrations of primer (1 uM), longer extension time (1 min) and two-step cycling (98C and 72C), theorizing that this should reduce chimera formation; however they didn't test any other protocols, so the importance of these details is unclear.

Many factors identified in other studies (such as template and primer concentrations, type of polymerase) could result in more or less cycles in plateau phase, and affect the degree of chimera formation in this way. But I would guess that if one stays in the exponential phase of PCR (good practice for many reasons), the higher processivity and lower error rate of Phusion or similar polymerases should give fewer artifacts.
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