Diversity Profiling FAQs

The following set of questions will help you navigate our Diversity Profiling service.

You can access the DivPro Sample Preparation Guide here.

How do I submit my samples to AGRF's DivPro service?

How much DNA do I need to submit?

I don’t have 10uL of 10ng/uL of DNA, can I still submit my samples?

How should I name my samples?

When will I find out if my samples have passed QC?

What happens if my samples do not pass the QC?

What happens if my samples are still inhibitory to PCR?

AGRF offers several PCR targets for Diversity Profiling – which one is right for me?

Will I be invoiced for samples that fail QC?

What does DivPro tell me about my sample?

Can I compare samples from one run with samples from a different run?

What kind of data do I receive from this service? And how do I download it?

My samples did not meet the minimum read guarantee; will AGRF repeat the sample/s?

Does the analysis pipeline use one or both reads?

How will rare species with low biomass or negative control samples behave in the DivPro service?

Can I submit a single sample?

I want to use negative control data to compare to my sample output, how can I do this?

Why are there organisms in my OTU results that aren’t from the same Kingdom as the PCR target?

How can I analyse my data?

How do I submit my samples to AGRF DivPro service?

Contact AGRF for a quote request using our online quote request wizard, or contact us. We can assist with pricing information and setting up your account so that you can submit your samples to this service.

 

How much DNA do I need to submit?

AGRF recommends submitting at least 100ng of gDNA in a 10uL volume (i.e. 10ng/uL). If you are requesting multiple targets to be amplified and sequenced from each sample, submit 10uL per target (e.g. for 3 targets, submit 30uL).

 

I don’t have 10uL of 10ng/uL of DNA, can I still submit my samples?

Yes, we can try to amplify any samples submitted regardless of DNA concentration and will let you know if they do not pass QC.

 

How should I name my samples?

For optimal labelling on the resulting bar and area charts, the AGRF recommends using intuitive sample names, limited to 7 characters.

 

When will I find out if my samples have passed QC?

AGRF will contact you after the initial PCR’s have been completed to let you know the QC results, as visualised on agarose gel.  Depending on when you submit your samples, this will vary between 1 – 3 weeks from when your samples are received.

 

What happens if my samples do not pass the QC?

AGRF will perform one repeat PCR using a dilution of your sample such as a 1:10 or 1:100 (depending on the concentration of the submitted material, and the likelihood of it containing PCR inhibitors such as humic acid).  In the event the samples do not amplify after this repeat attempt, we will be unable to continue further processing.  We do have available additional troubleshooting methods and it may be possible to investigate your result further and place the samples on to the following sequencing run.

 

What happens if my samples are still inhibitory to PCR?

If your samples still won’t amplify after dilution, AGRF offers a DNA clean-up service using the “Aurora” platform, which is a “sledgehammer” solution for removing PCR inhibitors. Contact us for pricing and further information.

 

AGRF offers several PCR targets for Diversity Profiling – which one is right for me?

Each target will give a different view of the same sample’s diversity. This is because during PCR amplification, some species amplify better than others, for that particular set of primers. While these targets are suitable for general overviews of a population, if you have a specific set of organisms that you are interested in, you should check the available literature to see if they are well represented by the target you choose, or if they have any bias that would affect your interpretation of the data.

 

Will I be invoiced for samples that fail QC?

Only gDNA samples that are sequenced will be invoiced. If you requested DNA extraction as part of the service, the DNA extraction will be charged for independently of the sequencing results.

 

My samples did not meet the minimum read guarantee; will AGRF repeat the sample/s?

We have a free repeat service for any sample that passes all pre-sequencing QC metrics but does not meet the minimum read guarantee.  Please request a rerun of your sample and we will organise this on the next available run using the supplied gDNA sample from your initial submission.

 
What does DivPro tell me about my sample?

There is a great deal of literature regarding the pros and cons of this type of assay, and what it can, and can’t tell you. The assay is not quantitative, and the community structure it reveals is only one possible view of the community. A different choice of amplicon, or DNA extraction procedure, might well reveal a different view of the community.

 
Can I compare samples from one run with samples from a different run?

Ideally, we would advise you to combine all samples that you want to compare into a single analysis, if you can. That avoids unnecessary variation, and ensures that all your samples are viewed in the same way by the analysis software, in particular the Chimera-checking aspects.

We understand that you might not always be able to run all of your samples together. In this case, we suggest that you contact us to request a combined re-analysis of your data, to ensure that optimal comparisons are being made. There is a charge for this service.

What kind of data do I receive from this service? And how do I download it?

You will receive “.fasta” files of the run output, as well as a table of the taxonomic units (OTUs) in your samples, and a chart of this data.

Once your data is available, you will receive an email notifying you that it can be downloaded from the client login area of our website.

My samples did not meet the minimum read guarantee; will AGRF repeat the sample/s?

We have a free repeat service for any sample that passes all pre-sequencing QC metrics but does not meet the minimum read guarantee. Please request a rerun of your sample and we will organise this on the next available run using the supplied gDNA sample from your initial submission.

Does the analysis pipeline use one or both reads?

Forward and reverse reads are merged at the start of the analysis pipeline. If the median assembly is > 65%, then the analysis continues with the merged reads. If the median assembly is less than 65%, then a single read approach is taken. The assembly metrics are included in the pdf of the results.

How will rare species with low biomass or negative control samples behave in the DivPro service?

The relative amplification efficiency for each bacterial species is a nonlinear function of the fraction that each of those taxa represent within a community or multispecies DNA template.  Consequently, the low-proportion taxa in a community are under-represented during PCR-based surveys and a large number of sequences might need to be processed to detect some of the bacterial taxa within the ‘rare biosphere’. The structure of microbial communities from PCR-based surveys is clearly biased against low abundant taxa with care required to decipher the complete extent of microbial diversity in nature.  Reference - http://www.ncbi.nlm.nih.gov/pubmed/22253843

Negative samples often yield a range of contaminating bacterial species, and these species are also seen in samples processed concomitantly.  The presence of contaminating sequences is greater in low-biomass samples than in high-biomass samples due to the self-normalising nature of PCR amplification. Reference - http://www.ncbi.nlm.nih.gov/pubmed/25387460

Can I submit a single sample?

With a single sample, if a sequence is only seen once, then it will be considered spurious and hence will be removed during the analysis. With multiple samples, the analysis takes into account sequences seen across samples and hence more low-abundance species will be retained.

I want to use negative control data to compare to my sample output, how can I do this?

It can be important to compare data from negative controls to your sample data, especially when your samples contain low target biomass. If you wish to do this, please take care to submit “blank” samples alongside your “test” samples. If you are submitting DNA to the service, you should use “blank” DNA extractions for this purpose. If the AGRF is providing you with a DNA extraction service, please think carefully about what you can submit that most accurately reflects a “blank” sample, eg unused swabs, filters, or collection tubes. The AGRF runs blank sequencing controls for internal purposes, however sequencing data from these do not reflect your input sample and will not be made available to you. Negative control samples will almost always generate sequencing output. There is a rich literature available on this type of artifact.

Why are there organisms in my OTU results that aren't from the same Kingdom as the PCR target?

You may recover sequences that match to animals and plants organellar and nuclear SSU rDNAs in your bacterial target (16S) results. This is because those targets share similar regions, due to the evolutionary history of life. For example, plastids, mitochondria, and other organelles contain genomes with one or more ribosomal RNA operons, which can co-amplify when using some 16S primers.

See the following examples:

www.ncbi.nlm.nih.gov/pubmed/23968645 www.ncbi.nlm.nih.gov/pmc/articles/PMC3008469

The presence of “off-target” reads is not uncommon, especially when significant amounts of DNA from other organisms is present, eg when analysing bacteria associated with eukaryotic hosts.

Minimising these off-target or “host” DNA products from amplifying can be difficult. Some primers work better than others at avoiding off-target amplification in particular types of samples (e.g. the 16S:V1-V3 service offered by AGRF will amplify plant chloroplasts/cyanobacterial 16S region very well, while the primers used in the 16S:V3-V4 service will not; www.nature.com/ismej/journal/v2/n4/fig_tab/ismej200797t2.html).

Beyond primer choice, it is possible to add a “blocking” primer into the primary PCR amplification, that will bind to host DNA and inhibit amplification (eg. www.earthmicrobiome.org/emp-standard-protocols/18s/). Please note that AGRF does not currently offer this as part of our DivPro service; contact us if you wish to provide such a blocker and have us include it in your amplification reactions.

Alternatively, you may choose to enrich your gDNA prior to submission using kits that can remove eukaryotic DNA from your purified extract, based on the presence of CpG methylation (e.g. www.neb.com/tools-and-resources/feature-articles/addressing-challenges-in-microbiome-dna-analysis) or differential lysis (www.molzym.com/products/next-generation-sequencing/ultra-deep-microbiome-prep). 

How can I analyse my data?

The most widely used marker gene pipelines are QIIME and mothur.

One Codex is a web-based tool that can analyse FASTQ data. Please note that it is currently set up primarily for shotgun sequencing rather than marker gene sequencing, and also currently uses human sequence as the host background, so will be less effective if used with non-human microbiomes.

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