Opinions on the best 16s and ITS primers & amplification protocols for unbiased microbiome analysis?

[Emerick Larkin]

I have and am still extracting (w/ MOBIO PowerSoil kit) DNA from the lettuce rhizosphere in preparation for conducting a microbial analysis of both the fungal and bacterial communities present on and within the lettuce roots. The lettuce is hydroponic so the microbial DNA levels will be low compared to the root cell DNA. I would like to do an unbiased amplification of both 16s (for bacterial community analysis) and ITS (for fungal community analysis) DNA, independently on each sample, for eventual sequencing on the MiSeq.  

Does anyone have any opinions on the best 16s and/or ITS primers and/or amplification protocols to fit these needs?

Some additional questions:

Should be doing real-time/qPCR on the DNA samples if I wish to do comparisons of the relative levels of different OTUs post-sequencing? My guess is no.

Should I be doing real-time/qPCR on the DNA samples if I wish to do comparisons of the relative levels of different OTUs post-sequencing? My guess is that it is not necessary.

What is the best way to be able to compare relative levels of fungal and bacterial OTU's from amplicons of the same sample (they have to be amplified separately right?) or is this not possible due to variations in the PCR/sequencing? 

Thanks in advance. 

[Xabier Vázquez Campos]

What kind of bias you want to avoid? Because even the extraction methods have bias. The PCR primers are well known to be biased and the sequencing primers won't give you the same exact results depending the region you amplify.

If you want the most standarised way to proceed go to the Earth Microbiome Project website and use whatever they say.

Amplicon sequencing doesn't give you absolute quantification but a relative composition of the taxa you are sequencing. Consider that you don't even have an uniform distribution of the copy number of the 16S, you can have between 1-15 copies per bacteria.

If you want exact cell numbers use fluorescence-based methods: flow cytometry, FISH, or manual counts under the microscope with SYBR or DAPI

[Abizar Lakdawalla]

This is a tough one. In general NGS data like from MiSeq is quite quantitative, it will give you a count of how many times a specific sequence occurs. Software on the Illumina BaseSpace gives you a quantitative distribution of bacterial families but not of species. But as mentioned by Xabier, quantitation at the species level is difficult unless you have calibrators. Another issue is the Greengenes database used as a reference is not without noise and annotation issues so some of the species level ID may be questionable.

For my curiosity, what are you trying to answer. That plant productivity is related to the types of microbiomes associated with the root system? 

BTW, Illumina has a library prep protocol that you could use for guidance.

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[Xabier Vázquez Campos]

Personally, I never use GG database if I can avoid it. It's quite abandoned and way less curated than Silva. For fungal ITS, use UNITE

On Thursday, 8 September 2016 13:02:57 UTC+10, Abizar Lakdawalla wrote:

This is a tough one. In general NGS data like from MiSeq is quite quantitative, it will give you a count of how many times a specific sequence occurs. Software on the Illumina BaseSpace gives you a quantitative distribution of bacterial families but not of species. But as mentioned by Xabier, quantitation at the species level is difficult unless you have calibrators. Another issue is the Greengenes database used as a reference is not without noise and annotation issues so some of the species level ID may be questionable.

For my curiosity, what are you trying to answer. That plant productivity is related to the types of microbiomes associated with the root system? 

BTW, Illumina has a library prep protocol that you could use for guidance.

On Wed, Sep 7, 2016 at 4:37 PM, Xabier Vázquez Campos <xvaz...@gmail.com> wrote:

What kind of bias you want to avoid? Because even the extraction methods have bias. The PCR primers are well known to be biased and the sequencing primers won't give you the same exact results depending the region you amplify.

If you want the most standarised way to proceed go to the Earth Microbiome Project website and use whatever they say.

Amplicon sequencing doesn't give you absolute quantification but a relative composition of the taxa you are sequencing. Consider that you don't even have an uniform distribution of the copy number of the 16S, you can have between 1-15 copies per bacteria.

If you want exact cell numbers use fluorescence-based methods: flow cytometry, FISH, or manual counts under the microscope with SYBR or DAPI


On Thursday, 8 September 2016 00:27:22 UTC+10, Emerick Larkin wrote:

I have and am still extracting (w/ MOBIO PowerSoil kit) DNA from the lettuce rhizosphere in preparation for conducting a microbial analysis of both the fungal and bacterial communities present on and within the lettuce roots. The lettuce is hydroponic so the microbial DNA levels will be low compared to the root cell DNA. I would like to do an unbiased amplification of both 16s (for bacterial community analysis) and ITS (for fungal community analysis) DNA, independently on each sample, for eventual sequencing on the MiSeq.  

Does anyone have any opinions on the best 16s and/or ITS primers and/or amplification protocols to fit these needs?

Some additional questions:

Should be doing real-time/qPCR on the DNA samples if I wish to do comparisons of the relative levels of different OTUs post-sequencing? My guess is no.

Should I be doing real-time/qPCR on the DNA samples if I wish to do comparisons of the relative levels of different OTUs post-sequencing? My guess is that it is not necessary.

What is the best way to be able to compare relative levels of fungal and bacterial OTU's from amplicons of the same sample (they have to be amplified separately right?) or is this not possible due to variations in the PCR/sequencing? 

Thanks in advance. 

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[Emerick Larkin]

It's not that I am necessarily trying to avoid bias for any particular microbe. I guess I am just looking for primers that have a particular degeneracy (coverage vs non-specific amplification tradeoff) so that they are as broadly nonspecific as possible without giving nonsense reads.

I am aware that every step has some bias one way or another, though. I have been looking at the EMP protocols and see that they have primers that have been modified to reduce bias for Crenarchaeota and Alphaproteobacteria. Are these primers designed for broad application across all environments? 

I am not looking for absolute quantification, just general a relative abundance comparison ( I realize that marker gene copy levels must also be taken into account for this). 

I do not yet know the basic structure of this community so antibody-cell-specific techniques like FISH or direct cell counting won't do me any good at this point in my research. 

I see that on the EMP protocol webpage they list only protocols for 16s and 18s rDNA amplification. I have received advice from professors at my university that ITS is a better region to target. Are 18s or ITS databases more developed? I don't think I can do both regions so I would like to choose the one which has better developed databases. 

Thanks for the feedback Xabier, it is appreciated. 

[Emerick Larkin]

Thank you for the input Abizar (cool name), this is the beginning of my research and right now I am simply trying to get a basic understanding of the microbiomes present in the rihzosphere of hydroponic lettuce. This is for eventual exploration of biocontrol methods of hydroponic pathogens and application into dual purpose wastewater reclamation and food production hydroponic systems for space colonies and sustainable urban centers on Earth. 

Do you agree with Xabier that the Silva database is better kept and more developed than the Green Genes database ?  

On Wednesday, September 7, 2016 at 11:02:57 PM UTC-4, Abizar Lakdawalla wrote:

This is a tough one. In general NGS data like from MiSeq is quite quantitative, it will give you a count of how many times a specific sequence occurs. Software on the Illumina BaseSpace gives you a quantitative distribution of bacterial families but not of species. But as mentioned by Xabier, quantitation at the species level is difficult unless you have calibrators. Another issue is the Greengenes database used as a reference is not without noise and annotation issues so some of the species level ID may be questionable.

For my curiosity, what are you trying to answer. That plant productivity is related to the types of microbiomes associated with the root system? 

BTW, Illumina has a library prep protocol that you could use for guidance.

On Wed, Sep 7, 2016 at 4:37 PM, Xabier Vázquez Campos <xvaz...@gmail.com> wrote:

What kind of bias you want to avoid? Because even the extraction methods have bias. The PCR primers are well known to be biased and the sequencing primers won't give you the same exact results depending the region you amplify.

If you want the most standarised way to proceed go to the Earth Microbiome Project website and use whatever they say.

Amplicon sequencing doesn't give you absolute quantification but a relative composition of the taxa you are sequencing. Consider that you don't even have an uniform distribution of the copy number of the 16S, you can have between 1-15 copies per bacteria.

If you want exact cell numbers use fluorescence-based methods: flow cytometry, FISH, or manual counts under the microscope with SYBR or DAPI


On Thursday, 8 September 2016 00:27:22 UTC+10, Emerick Larkin wrote:

I have and am still extracting (w/ MOBIO PowerSoil kit) DNA from the lettuce rhizosphere in preparation for conducting a microbial analysis of both the fungal and bacterial communities present on and within the lettuce roots. The lettuce is hydroponic so the microbial DNA levels will be low compared to the root cell DNA. I would like to do an unbiased amplification of both 16s (for bacterial community analysis) and ITS (for fungal community analysis) DNA, independently on each sample, for eventual sequencing on the MiSeq.  

Does anyone have any opinions on the best 16s and/or ITS primers and/or amplification protocols to fit these needs?

Some additional questions:

Should be doing real-time/qPCR on the DNA samples if I wish to do comparisons of the relative levels of different OTUs post-sequencing? My guess is no.

Should I be doing real-time/qPCR on the DNA samples if I wish to do comparisons of the relative levels of different OTUs post-sequencing? My guess is that it is not necessary.

What is the best way to be able to compare relative levels of fungal and bacterial OTU's from amplicons of the same sample (they have to be amplified separately right?) or is this not possible due to variations in the PCR/sequencing? 

Thanks in advance. 

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[Abizar Lakdawalla]

Thanks Emerick, interesting project. 

Not sure it matters which database as most likely you will be using an already available piece of software.

Good luck with the project!

(One more link of a service provider who can do the work (I have no connection with these folks). http://www.secondgenome.com/solutions/services/)

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[Xabier Vázquez-Campos]

Emerick,
The EMP is focused on soil, so the primers are optimised... for soils; saying that, consider that soil is the with highest microbial diversity and cell counts, and it's still one of the most complicated systems despite all the people working on it. So I'd go with their primers.

Regarding 18S/ITS... what do you want to 'see'? In general, ITS primers target fungi, while 18S target any Eukarya. The problem with the 18S, at least in fungi, is that lacks resolution. Fungal 18S is very conserved and unless you do high-rank phylogenies, it does not tell you much (but you'll know if you got any 'protozoa' and such). Also, you may have issues amplifying 'rogue' plant material from your DNA extractions.
On the other hand, ITS is anything but conserved... it has so much variability that is not phylogenetically informative, but is a very good barcode and fungal databases are pretty well established. Due to the variability, the data processing is a bit different in a couple of steps compared to 16S (at least it's what I read as I never did ITS amplicon seq)

[Emerick Larkin]

Thank you Xabier, great input! I am not interested in any other microbes other than bacteria and fungi at this point and I also want to avoid amplification of plant DNA. ITS it is then! 

Now I will just have to find some suitable ITS primers. 

[Xabier Vázquez-Campos]

I've used the classical ITS5/ITS4 primer set for normal PCR (White et al 1990) that spans the whole ITS region but pretty sure that the EMP guys have something better.