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Old 01-12-2012, 07:41 AM   #1
ramsemm
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Default V4 Region Amplification and Sequencing via MiSeq

Hi Everyone,

I am new to the forum and am hoping to find some help regarding primer design and sequencing for conducting 16S metagenomics studies using the Illumina MiSeq.

I have previously worked with preparing samples for and analyzing 454 data and am switching to using an Illumina MiSeq. Currently, I am interested in amplifying the V4 region of 16S and have the primer sequences targeting that region. However, the custom amplicons from Illumina generated using DesignStudio (which include their adapters) from my understanding are only able to be based on the human genome rather than bacterial. So, I am trying to understand how to go about designing the primers and linkers needed for amplifying my region of interest that will include the adapter regions needed to match those on the Illumina flow cell.

In a recent paper by Caporaso et al. (2011), there is a figure depicting the initial and sequencing primers with the forward primer having an Illumina adapter, linker, and primer sequence and the reverse having all of these along with the barcode sequence. This is what I would like to do, but I do not understand how to get the adapter sequence to match the Illumina-specific adapters located on their flow cell. I have a feeling it is just that I am new to this entire process and am missing something. So, I thought I would check with the forum to see if anyone had any suggestions.

Thank you all in advance!
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Old 01-12-2012, 07:55 AM   #2
kmcarr
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ramsemm,

The Sequences you need to know are shown in the Caporaso paper you cited. The bases shown in green at the 5' ends of the forward and reverse PCR primers (Figure 1, panel 2) are the sequences required for annealing to the flow cell and doing the bridged amplification. Seeing as how Caporaso's primers target the V4 region you could simply use those sequences as is (ordering as many barcoded reverse primers as necessary).
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Old 01-12-2012, 08:00 AM   #3
ramsemm
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Thanks kmcarr!

I wondered if that was okay, but I thought it might be just too simple. As far as determining the barcodes, does each company (like Illumina) have their own preferred barcodes that they know work best with their system? Or particular barcodes that they suggest to use together or not together?

Thank you again for the help! I am just getting started with all of this. In the past, my samples were sent off, and I did not get to understand the specifics of design. Just trying to make sure I understand it all correctly this time around.
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Old 01-12-2012, 08:55 AM   #4
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Yes. There are Illumina recommended barcode sequences which you should be able to get by asking your local Illumina rep (you can also find them with a quick Google search).
We are also looking to do something similar - moving from 454 to MiSeq. We are hoping to try and use a dual indexing approach appending the Nextera barcode sequences to both the forward and reverse primers. It means we should be able to multiplex 96 samples with just 20 primers. The Nextera sequences are in the Nextera manual from the Illumina website.
You also have to use custom sequencing primers (the same as those needed to amplify V4 but without the Illumina adapters appended) otherwise your first bases will be primer and low diversity, which is not good.

There's also a application note on the Illumina website on V4 sequencing if you haven't already seen that.
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Old 04-23-2012, 07:57 AM   #5
jwcimug
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Hello,

I am also interested in sequencing V4 on MiSeq for 16S studies. Can anyone recommend a good service provider to do this?

Thank you!
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Old 01-09-2013, 10:08 AM   #6
costamc
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Hi,
we are also moving from 454 to Illumina MiSeq. Does anybody have any suggestion on which region of the 16S rRNA gene to use for the new chemistry that amplifies 2x250bp? I was hoping to use the PE sequencing and get an overlap of around 50bp.
Thanks in advance.
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Old 01-09-2013, 11:11 AM   #7
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I would suggest reading
http://nar.oxfordjournals.org/conten.../31/nar.gks808

The ARB group put this one out last year. Great in-silico evaluation of a number of primer pairs for this topic. We ended up going for one of the primer pairs that should cover archaea and bacteria relatively equally.
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Old 01-10-2013, 06:00 AM   #8
MLog
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Hi!
I'm also thinking about sequencing of 16S amplicons using protocol from Caporaso et al. (2011). But how to deal with the low-complexity issue? Illumina recommends to add the PhiX library (and I thought about adding some of my gDNA libraries that I need to be sequenced). But if I understand correctly, the protocol by Caporaso et al. requires custom sequencing primer. If so, it will not work with phiX or any other library prepared with standard Truseq adapters.
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Old 01-10-2013, 06:55 AM   #9
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Quote:
Originally Posted by MLog View Post
Hi!
I'm also thinking about sequencing of 16S amplicons using protocol from Caporaso et al. (2011). But how to deal with the low-complexity issue? Illumina recommends to add the PhiX library (and I thought about adding some of my gDNA libraries that I need to be sequenced). But if I understand correctly, the protocol by Caporaso et al. requires custom sequencing primer. If so, it will not work with phiX or any other library prepared with standard Truseq adapters.
You spike in the custom primer into the primer mix at 0.5ÁM. The primer mix will contain both the PhiX and custom primer.
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Old 01-14-2013, 10:34 AM   #10
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Does anyone know what the primer pad part of the Caporaso fusion primers does? I can't seem to figure out what the definition of a primer pad is!
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Old 01-14-2013, 12:06 PM   #11
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Quote:
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Does anyone know what the primer pad part of the Caporaso fusion primers does? I can't seem to figure out what the definition of a primer pad is!
I was thinking about this just today! I'm not sure but my version is that this is a sequence introduced for better performance of custom sequencing primer. The major part of this primer corresponds to 16S-specific sequence, which is degenerate, and not very long (only 20 nt). Thus these extra 10 letters are need to "reinforce" the primer, improve its annealing.
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Old 01-14-2013, 12:12 PM   #12
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Quote:
Originally Posted by TonyBrooks View Post
You spike in the custom primer into the primer mix at 0.5ÁM. The primer mix will contain both the PhiX and custom primer.
Thanks, TonyBrooks!
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Old 01-14-2013, 01:39 PM   #13
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Quote:
Originally Posted by microgirl123 View Post
Does anyone know what the primer pad part of the Caporaso fusion primers does? I can't seem to figure out what the definition of a primer pad is!
It provides a "pad" for the primers to anneal for the sequencing versus amplification primers. Hard to explain in perfect detail in text but if you diagram the amplicon and draw where each primer starts and ends, the role of the pad is a bit easier to see. The primer pad is the same as the 5'-end of the sequencing primers (read 1 and read 2) and the same as the 3' end of the index read primer.

Note that the 2011 Caporaso PNAS paper may have a typo in the sequences shown in Figure 1. Unless I'm missing something, the Read 2 sequencing primer and the Index read primer are not compatible with each other. One of them has a typo and I think the Index sequence is correct and matches the primer sequence list provided in supplemental material in that paper or one of his other recent papers.
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Old 01-15-2013, 01:21 AM   #14
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Quote:
Originally Posted by csquared View Post
It provides a "pad" for the primers to anneal for the sequencing versus amplification primers. Hard to explain in perfect detail in text but if you diagram the amplicon and draw where each primer starts and ends, the role of the pad is a bit easier to see. The primer pad is the same as the 5'-end of the sequencing primers (read 1 and read 2) and the same as the 3' end of the index read primer.
It's due to the relatively high Tm on the MiSeq (65C).
The padding allows for the design of a longer custom read primer (and higher Tm) without having to extend sequence into the P5/P7 regions.
We confirmed this with the author.
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Old 01-15-2013, 02:27 AM   #15
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Quote:
Originally Posted by TonyBrooks View Post
It's due to the relatively high Tm on the MiSeq (65C).
The padding allows for the design of a longer custom read primer (and higher Tm) without having to extend sequence into the P5/P7 regions.
We confirmed this with the author.
Hi,

I am also trying to make a design for MiSeq amplicon sequencing. In the supplementary file of the paper it is written that "The amplification and sequencing primers additionally contain a new pad region to avoid primer-dimer formation with the modified adapter."

Is use of primer pad because of Tm or primer-dimer formation in the long amplicon primers? Is it necessary to design new primer pads when using different 16S rRNA primers? Thank you for your help.
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Old 01-21-2013, 05:35 AM   #16
liepa
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Quote:
Originally Posted by csquared View Post
It provides a "pad" for the primers to anneal for the sequencing versus amplification primers. Hard to explain in perfect detail in text but if you diagram the amplicon and draw where each primer starts and ends, the role of the pad is a bit easier to see. The primer pad is the same as the 5'-end of the sequencing primers (read 1 and read 2) and the same as the 3' end of the index read primer.

Note that the 2011 Caporaso PNAS paper may have a typo in the sequences shown in Figure 1. Unless I'm missing something, the Read 2 sequencing primer and the Index read primer are not compatible with each other. One of them has a typo and I think the Index sequence is correct and matches the primer sequence list provided in supplemental material in that paper or one of his other recent papers.
Has the "primer pad" to be adjusted for the amplified 16S region?
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Old 07-02-2013, 03:58 AM   #17
Kumari Richa
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Default Primers for V4 region

Hi,

I am new to the forum and extremely new to Illumina. I am planning to amplify the V4 region of Bacterial 16S rRNA. I look forward to learn very basic protocol of the PCR amplification, PCR program for the same. Also about what primer sets I can use? Are Adapters included in the primer? What is a barcode ? Is it employed during the pcr amplification or during the sequencing? How to choose it ?
I want to just amplify the desired fragment and send it for sequencing. Please help.
Thanks
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Old 07-02-2013, 04:17 AM   #18
GenoMax
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Quote:
Originally Posted by Kumari Richa View Post
Hi,

I am new to the forum and extremely new to Illumina. I am planning to amplify the V4 region of Bacterial 16S rRNA. I look forward to learn very basic protocol of the PCR amplification, PCR program for the same. Also about what primer sets I can use? Are Adapters included in the primer? What is a barcode ? Is it employed during the pcr amplification or during the sequencing? How to choose it ?
I want to just amplify the desired fragment and send it for sequencing. Please help.
Thanks
Here is a link to the App Note that Illumina has for 16S sequencing: http://res.illumina.com/documents/pr..._miseq_16s.pdf
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Old 07-02-2013, 04:46 AM   #19
kmcarr
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Quote:
Originally Posted by Kumari Richa View Post
Hi,

I am new to the forum and extremely new to Illumina. I am planning to amplify the V4 region of Bacterial 16S rRNA. I look forward to learn very basic protocol of the PCR amplification, PCR program for the same. Also about what primer sets I can use? Are Adapters included in the primer? What is a barcode ? Is it employed during the pcr amplification or during the sequencing? How to choose it ?
I want to just amplify the desired fragment and send it for sequencing. Please help.
Thanks
If you haven't done so already read the Caporaso (2011) paper (see References in he Illumina App Note that GenoMax linked to above.) This paper is from Rob Knight's lab, the folks that wrote QIIME, one of the more popular tools for analyzing 16S sequence data.

There is also this detailed protocol from Patrick Schloss' lab posted here. The Schloss lab wrote mothur, another popular tool for 16S sequence analysis.
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Old 07-02-2013, 07:11 AM   #20
scotto
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We have successfully run v4 16s samples on the MiSeq using the Caporaso method and the Bioo Scientific product, which is just a kitted version of the Caporaso method. Both have worked with MiSeq Reporter, but our Bioinformatics group have also run their own analyses. As a service provider in a core facility I prefer the convenience of the kit, but that's just my preference. We have been using about a 10% phiX spike in with the newest version of the MiSeq software.
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