[liepa]

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?

[Kumari Richa]

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

[GenoMax]

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

[kmcarr]

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.

[scotto]

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.

[fozrun]

Dual Index method published by Pat Schloss

Pat Schloss has published a dual index method for v4 in AEM.

http://aem.asm.org/content/early/2013/06/17/AEM.01043-13.abstract

Interstingly he gets better quality with the shorter v4 amplicon than the longer v3-v4.

I've also designed a v3-v4 dual index assay based on the ARB recommendations for primers and it works quite well.

Bob

[Genohub]

I'm interested in your V3-V4 design. Is it published somewhere? Anyone have success using these primers for other variable 16S domains?

[fozrun]

Dual Index method

No it's not published yet, but it will be submitted as part of a paper in about three weeks. PM and I can send you the details.

We used the Illumina barcodes for the first 20 primers (8 fwd, 12 rev) and then designed another two sets using the Faircloth & Glenn edit_metric 4 sequence tags. We also designed a set for archaea that we are testing next week.

[epistatic]

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.

Did you run human genomic DNA as input? If so, how much do you recommend as input? I took a look at Bioo's kit and they used 50 ng of bacterial DNA as input. Understand this for straight bacterial ID but would like to use for metagenomic screen.

Thank you!

[scotto]

Did you run human genomic DNA as input? If so, how much do you recommend as input? I took a look at Bioo's kit and they used 50 ng of bacterial DNA as input. Understand this for straight bacterial ID but would like to use for metagenomic screen.

Thank you!

We used it for microbial metagenomics starting with 50 ng of DNA from a fecal extract. We didn't use any human input. We spiked in about 5-10% phiX to help the basecaller overcome the low diversity.

[Genohub]

entire 16S region

Has anyone designed primer sets to cover the entire 16S region (not just v4)? I would be really interested in that !

- Genohub

[m232]

Hi, I am new in the Forum and Illumina Miseq and I have some basic doubst that I would like to solve. I am planning on characterizing the taxonomic diversity in 40 of my samples, and amplify the V4 region of the 16S rRNA gene (the samples come from an algae and the associated bacteria that live in symbiosis on it). So I assume that the diversity within my samples will be quite low.

- As I am new, I don´t want to run all the samples at once and I was thinking to do 2 runs in Miseq, what will means 20 samples per run. But I am not sure if that will be two low.

- For what I read in the forums, I will also have to spike with phiX. Illumina recommended 5% although I have read in some posts that that might be a bit low especially for my low diversity samples.

- Also, does anybody know how many samples can be sequenced at once in the MISeq in order to be able to calculate betha diversity. I guess 20-40 samples is quite low as I see that some people run up to 400 samples at once.

Thank you!

[Genohub]

What version of the MiSeq software are you using? The latest version has some corrections that allow you to add only 5-10% PhiX. Before the correction, it was more like 35-40% PhiX. Does anyone understand the details of this diversity "correction"?

The number of samples/lane depends on the numbers of reads you need per sample to characterize your taxonomic diversity. Check our the Caporaso paper for recommendations.

- Genohub

[Nirajlincoln]

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!

Hi, I am also planning to do 16S amplicon or rRNA sequencing using on Illumina MiSeq platform for 70 fecal samples. Can you please advise me shich one is cheapest method because I want to analysis QIIME, functional and pathways etc. Can you please also advise which one is cheapest in terms of costs for 70 samples?

[Genohub]

16S V4 Providers

Hi, I am also planning to do 16S amplicon or rRNA sequencing using on Illumina MiSeq platform for 70 fecal samples. Can you please advise me shich one is cheapest method because I want to analysis QIIME, functional and pathways etc. Can you please also advise which one is cheapest in terms of costs for 70 samples?

Are you planning on doing the library preparation and sequencing yourself? Several providers on Genohub.com offer 16S V4 services. Are you interested in looking at several domains or just one domain? In either case I recommend single PCR sets where your primers contain regions variable domain flanking + seq primer regions.

- Genohub