best primers for 16S amplicon sequencing

dca · 04-26-2010, 12:07 PM

Hello All
We are starting a metagenomics project to identify mixed bacterial populations in various biological specimens. Any suggestions for robust 16S primers?
Ideally, something that has been successfully used with the GS-FLX platform and contains the necessary key, A/B and possibly MID sequences.
Thanks!
David

kmcarr · 04-26-2010, 12:42 PM

David,

Have a look at what the folks at the Ribosomal Database Project have done with multiplexed primer design for 16S amplicon sequencing:

http://pyro.cme.msu.edu/pyro/help.jsp

greigite · 05-14-2010, 03:33 PM

Check out the papers from Mitch Sogin's group in woods hole- lots and lots of info on 16s amplicon sequencing using 454.

pmiguel · 05-26-2010, 05:00 AM

Quote:

Originally Posted by kmcarr

David,

Have a look at what the folks at the Ribosomal Database Project have done with multiplexed primer design for 16S amplicon sequencing:

http://pyro.cme.msu.edu/pyro/help.jsp

The designs here look optimized for GS-FLX standard chemistry. (~210 base amplicons and 6 or 8 base barcodes). Anyone done a redesign to take advantage of longer Titanium reads?

--
Phillip

cliffbeall · 07-29-2010, 09:49 AM

The HMP DACC has some info on primers and bar codes that were used for titanium 454 sequencing - http://www.hmpdacc.org/sops.php

They were looking at V1-V3 and V3-V5 regions.

Edit: They reorganized their web site, the proper url is: http://www.hmpdacc.org/tools_protocols.php#sequencing

v_kisand · 09-16-2010, 05:33 AM

And also check
Hong, S.-H, Bunge, J., Leslin, C., Jeon, S-O., and Epstein, S. S. (2009). Polymerase chain reaction primers miss half of rRNA microbial diversity. ISME Journal (advanced on-line publication doi: 10.1038/ismej.2009.89).

http://www.nature.com/ismej/journal/...ej200989a.html

Palecomic · 09-17-2010, 08:13 AM

For longer barcodes for titanium, Fierer 2008 PNAS has a good set http://www.pnas.org/content/early/2008/11/12/0807920105 we're using these with the human microbiome project primers (their barcodes are a couple of different lengths, I prefer Fierer's as the length is standard).

SeQnewby · 08-30-2012, 03:18 AM

Hi everyone

I am currently busy with a new metagenomics project that requires 16S and 18S rRNA pyrosequencing. However our new sequencer, the GS junior, I am told uses different adapter sequences to the FLX titanium etc. Does anyone know where I might find a tried and tested set? Full sequences. Also the RDP database gives a list of 8 base and 6 base barcodes, however our sequence technician insists on using roche's 10 base barcodes making the primers long without actually attaching the specific 16/18S primer....And our normal 16S primers that amplify the V3 region of the 16S forms hairpins with the adapter sequencing and causes mayor primer dimers...

help please please....

HMorrison · 09-07-2012, 01:00 PM

Quote:

Originally Posted by greigite

Check out the papers from Mitch Sogin's group in woods hole- lots and lots of info on 16s amplicon sequencing using 454.

We've finally got a publication out that details v6v4 Ti amplification and one in press for v3v5. Better than the HMP (single primer) versions.

Filkins LM, Hampton TH, Gifford AH, Gross MJ, Hogan DA, Sogin ML, Morrison HG, Paster BJ, O'Toole GA.
J Bacteriol. 2012 Sep;194(17):4709-17. Epub 2012 Jun 29.

and

Marteinsson, V., Runarsson, A., Stefansson, A., Thorsteinsson, T., Johannesson, T., Magnusson, S., Reynisson, E., Einarsson, B., Wade, N.*, Morrison, H. and E. Gaidos [corresponding author], A pervasive microbial community in waters under the Vatnajokull ice cap, Iceland, The International Society for Microbial Ecology Journal, in press

Still using 5 nt barcodes.

Palecomic · 09-11-2012, 10:49 AM

The guys at Silva (bigger and better aligned 16S database than the other alternatives) have done an exercise checking out all the 16S primers they could get their hands on.

Paper published here http://nar.oxfordjournals.org/conten...ar.gks808.full

We have adapted versions of the HMP V3-V5 primers here http://www.plosone.org/article/info%...l.pone.0032543 which perform well and using the criteria in Silva paper are both greater than 90% coverage for bacteria.

More important in some ways is knowing how the primers you pick perform with your particular sample type. We know ours aren't so hot on Mycobacteria for example, so for projects where that's an issue we can swap them out or use specific assays for those bugs within the community.

Also, it's not always about phylogenetic coverage - some great coverage primers give rubbish yields in PCR, so you have to deal with large numbers of samples dropping out.

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04-26-2010, 12:07 PM	#1
dca Junior Member Location: Canada Join Date: Apr 2010 Posts: 4	best primers for 16S amplicon sequencing Hello All We are starting a metagenomics project to identify mixed bacterial populations in various biological specimens. Any suggestions for robust 16S primers? Ideally, something that has been successfully used with the GS-FLX platform and contains the necessary key, A/B and possibly MID sequences. Thanks! David

07-29-2010, 09:49 AM	#5
cliffbeall Senior Member Location: Ohio Join Date: Jan 2010 Posts: 144	Titanium primers The HMP DACC has some info on primers and bar codes that were used for titanium 454 sequencing - http://www.hmpdacc.org/sops.php They were looking at V1-V3 and V3-V5 regions. Edit: They reorganized their web site, the proper url is: http://www.hmpdacc.org/tools_protocols.php#sequencing Last edited by cliffbeall; 09-16-2010 at 08:44 AM. Reason: Change in URL

08-30-2012, 03:18 AM	#8
SeQnewby Junior Member Location: South Africa Join Date: Aug 2012 Posts: 3	primers for GS Junior Hi everyone I am currently busy with a new metagenomics project that requires 16S and 18S rRNA pyrosequencing. However our new sequencer, the GS junior, I am told uses different adapter sequences to the FLX titanium etc. Does anyone know where I might find a tried and tested set? Full sequences. Also the RDP database gives a list of 8 base and 6 base barcodes, however our sequence technician insists on using roche's 10 base barcodes making the primers long without actually attaching the specific 16/18S primer....And our normal 16S primers that amplify the V3 region of the 16S forms hairpins with the adapter sequencing and causes mayor primer dimers... help please please....

04-26-2010, 12:42 PM	#2
kmcarr Senior Member Location: USA, Midwest Join Date: May 2008 Posts: 1,178	David, Have a look at what the folks at the Ribosomal Database Project have done with multiplexed primer design for 16S amplicon sequencing: http://pyro.cme.msu.edu/pyro/help.jsp

05-14-2010, 03:33 PM	#3
greigite Senior Member Location: Cambridge, MA Join Date: Mar 2009 Posts: 141	Check out the papers from Mitch Sogin's group in woods hole- lots and lots of info on 16s amplicon sequencing using 454.

09-16-2010, 05:33 AM	#6
v_kisand Member Location: Eesti Join Date: Jan 2009 Posts: 37	And also check Hong, S.-H, Bunge, J., Leslin, C., Jeon, S-O., and Epstein, S. S. (2009). Polymerase chain reaction primers miss half of rRNA microbial diversity. ISME Journal (advanced on-line publication doi: 10.1038/ismej.2009.89). http://www.nature.com/ismej/journal/...ej200989a.html

09-17-2010, 08:13 AM	#7
Palecomic Member Location: London Join Date: Aug 2010 Posts: 34	For longer barcodes for titanium, Fierer 2008 PNAS has a good set http://www.pnas.org/content/early/2008/11/12/0807920105 we're using these with the human microbiome project primers (their barcodes are a couple of different lengths, I prefer Fierer's as the length is standard).

09-11-2012, 10:49 AM	#10
Palecomic Member Location: London Join Date: Aug 2010 Posts: 34	The guys at Silva (bigger and better aligned 16S database than the other alternatives) have done an exercise checking out all the 16S primers they could get their hands on. Paper published here http://nar.oxfordjournals.org/conten...ar.gks808.full We have adapted versions of the HMP V3-V5 primers here http://www.plosone.org/article/info%...l.pone.0032543 which perform well and using the criteria in Silva paper are both greater than 90% coverage for bacteria. More important in some ways is knowing how the primers you pick perform with your particular sample type. We know ours aren't so hot on Mycobacteria for example, so for projects where that's an issue we can swap them out or use specific assays for those bugs within the community. Also, it's not always about phylogenetic coverage - some great coverage primers give rubbish yields in PCR, so you have to deal with large numbers of samples dropping out.