Frequently Asked Questions

Sequencing 101

What is Next Generation Sequencing?

Next Generation Sequencing (NGS) is a technology that can sequence millions of pieces of DNA in parallel in rapid fashion. Key aspects of NGS is that it has a low error rate, takes about 2 days to finish, and can sequence individual DNA segments that are 600 base pairs long.

What is an amplicon?

A short segment of DNA duplicated over and over, i.e. amplified, via PCR. In metabarcoding, amplicons are the segments of DNA of interest that we sequence.

What is an OTU?

OTU stands for “Operational Taxonomic Unit”. OTUs are clusters of sequences that are relatively similar and may represent multiple species. Since a single OTU can be multiple species, they are referred to as OTUs rather than species.

What is a primer?

A primer is a short section of DNA that has been synthesized. Its purpose is to bind to DNA segments during PCR and initiate amplification. Primers are used in pairs, one at the start of the DNA segment and one at the end.

What is BLAST?

BLAST is a technique that can be used to match a particular DNA sequence with sequences in a reference library. BLASTing allows one to figure out what species in the reference database share the same sequence as the target sequence.

What is quantitative PCR or qPCR?

qPCR is similar to regular PCR, but with each cycle the qPCR machine quantifies the amount of fluorescence that is generated by a probe that attaches to a specific sequence of DNA. The technique specializes in providing an absolute count of the number of copies of a sequence initially in a solution and can be made so that it is species specific. It is used mostly to detect the presence (or suggest the absence) of a single species and/or provide an absolute abundance of its DNA.

What is eDNA?

eDNA stands for “environmental DNA”. It is simply DNA found in the environment, i.e. outside of organisms. The DNA might be in sloughed off cells or be DNA that is bound to other substances like clays or organic particles. Empirically, for water, we call any DNA that we get on a filter to be eDNA, though it also includes DNA still in organisms like algae.

What is metabarcoding?

Metabarcoding is a technique to quantify the abundance of different species in mixtures. It uses PCR and primers that amplify a particular short region DNA for a large number of species at once. The “barcode” of metabarcoding is the species’ unique sequence for that region that acts as a barcode to identify it.

JonahAnimal

What primer pairs do you use?

For diet, we typically use trnL primers for plants, ITS primers for fungi, COI primers for insects, and 12S primers for vertebrates.

For parasites, we typically use 18S primers.

For pollen, we use the same trnL primers as for plants in diet.

For microbiomes, we use 16S primers for bacteria and ITS primers for fungi.

Upon consultation, we can use other primers to help quantify other taxonomic groups.

Can you determine what lichens my animal is eating?

Yes. We can either sequence for the alga or the fungus of the lichen (or both).

What is the best way to sample fecal material?

We send 20 mL scintillation vials. If you fill these half-way with sample, that will be more than enough sample. How you get the fecal material into there is up to you. For large fecals, the narrow end of a disposable spoon works well. For small samples, tweezers.

Do I need to know exactly how many samples I will be submitting ahead of time?

No, but a close estimate is helpful so that we can send you approximately the right number of vials.

Can I send you samples in other containers such as plastic bags?

In order to reduce the amount of cross-contamination and minimize sample mix-ups, using our supplied vials is best.

What if I am sending swabs and they don’t fit?

Clip them.

Should I write more information on the vial?

As long as our label and barcode are not impacted, you can add more labels or writing. If you fill out the datasheets correctly, we won’t need it. Typically we ignore any extra writing on a vial.

Are those labels safe for the fridge or freezer?

The labels are good down to -40°C.

Should I homogenize fecal samples?

DNA is pretty well evenly distributed in fecal samples. If multiple samples are being combined, these should be homogenized as much as possible to ensure representativity

Should I run replicates?

Scientifically, our general rule of thumb is to maximize statistical power. That means run as many independent samples as possible. Once the number of independent samples is maximized, it then makes sense to begin to replicate. How many replicates will be is hard to say. Every case is unique.

Should I keep duplicates?

We will archive samples until they are successfully sequenced. Samples are minimally handled and our techniques minimize the likelihood loss or contamination. Once we deliver samples and payment is approved, we typically will maintain samples for a few months afterwards, but unless directed, will not maintain samples indefinitely.

What if I cannot freeze the samples in the field? How do I preserve them?

Either dry them by adding silica gel to the vial, or preserve them with an ethanol solution. We can also recover plant DNA from air-dried samples.

Where and how do I ship my samples?
For most customers, the insulated shipper in which we sent vials to you initially can be used for returning samples.
Ship to:
Jonah Ventures
1600 Range Street Suite 201
Boulder, CO 80301
(785) 409-1655

Make sure to email us tracking information (info@jonahventures.com).

For frozen samples in an insulated shipper, 2-day express is usually sufficient. Fed Ex and UPS are typically better than USPS for shipping.

How many PCR replicates do you normally run?
Most animal samples are run with one PCR replicate. We can run samples with triplicate PCR pooled or unpooled. Contact us for pricing.
Can I send vouchers for potential diet items?
For any species not currently in our database, you can send a voucher for us to sequence and add to our database. For plants, just send a portion of a leaf in one of our vials. We’ll sequence it and add it to the database. Coordinate with us before doing this, though.

JonahPlant

What primer pairs do you use?

We use 16S primers for bacteria and ITS primers for fungi.

Upon consultation, we can use other primers to help quantify other taxonomic groups.

Where do I ship samples?
For most customers, the insulated shipper in which we sent vials to you initially can be used for returning samples.
Ship to:
Jonah Ventures
1600 Range Street Suite 201
Boulder, CO 80301
(785) 409-1655

Make sure to email us tracking information (info@jonahventures.com).

For frozen samples in an insulated shipper, 2-day express is usually sufficient. Fed Ex and UPS are typically better than USPS for shipping.

JonahWater

What is in the eDNA sampling kits?
Our eDNA sampling kits consist of a sterile 60 mL syringe, a 60 mL barcoded cup that contains a desiccant pack and a 1 µm filter.
What is the sampling protocol for eDNA?

At your site, sample 60 mL of water with the syringe. Attach the syringe filter and push water through. Repeat until you cannot push any more water through. This might be 60 mL in turbid water to 500 mL in clear water. Shake any water off the filter and replace it in the cup with the desiccant.

Are there any aids in pushing water through the syringe? My hand is starting to hurt.

You don’t have to push too hard in the end. The last few mL don’t help that much. But, when you have a lot of samples to push through in a day, sometimes your hand can get sore. We had that happen to us once. See here on how to prevent this.

 

Can we use filters with different pore sizes?
Our typical kit includes a filter with 1µm pores. For some specialized questions, filters with different pore sizes are helpful. We can advise you if this is the case.
How should samples be stored?
Desiccation at room temperature is sufficient to stabilize DNA. Samples can additionally be frozen if desired before sending them in to be analyzed.
Do we need different kits for NGS and qPCR?
Nope.
What species can you detect with qPCR?
Contact us for a list.
What primer pairs do you use for NGS?

We primarily use 16S primers for bacteria, 23S for phytoplankton, COI for zooplankton, COI for macroinvertebrates, 12S primers for fish and other vertebrates.

Upon consultation, we can use other primers to help quantify other taxonomic groups.

Where and how do I ship my samples?
Ship to:
Jonah Ventures
1600 Range Street Suite 201
Boulder, CO 80301
(785) 409-1655

Make sure to email us tracking information (info@jonahventures.com).

We typically recommend 2-day express. Fed Ex and UPS are typically better than USPS for shipping.

Sending cups in an uninsulated box is fine.

NGS Reports

The sample output says that my sample has DNA from a plant/insect/fish that at my site. Why?

Sometimes, the species actually is there. Often it isn’t, but more than likely some species closely related to species in the listed genus is found at your site. Our database of reference sequences is not all-encompassing and we cannot identify every species (yet).

Why do I not have data from some of my samples?

Some samples just do not amplify well – either do an insufficient amount of plant DNA in the sample or because it was difficult for us to get sufficiently clean DNA. We do our best, but we cannot guarantee data from 100% of samples. We examine all output and work with clients to see if resequencing is appropriate.

If the match is <100% can it still be a species in that genus?

Yes, but it is less likely to be a species in that genus. The OTU is based on a consensus sequence that represents a most probable sequence and a number of similar sequences that might differ in, say, 1 or 2 base pairs. So, multiple species can be represented by the consensus sequence, even if individuals of the different species do not have exactly the same sequence.

What if there is no match between an OTU ID and anything in the library?

If there is no match in the library, this means that no species that have been sampled match the consensus sequences at the 97% level. Generally, this is reported as an “unknown” in a paper, but more work can be done to narrow down what species they might represent. First, we’ll supply a “fasta” file, which has the actual consensus sequences. You can go to the NCBI website and look to see what species have the closest match and try to interpret that in the context of your samples. Or, you can collect leaves of species that are likely to match that unknown. We’ll sequence these and rerun the bioinformatics to see if the unknowns now match.

I sent plant samples to have run for the reference library. How do I interpret these data?

We sequence these samples similar to how we sequence diet samples. As such, you’ll see a percentage of reads assigned to each OTU. Often more than one OTU will have sequences. In short, the OTU that is assigned to this sample can now be interpreted to include this species if the OTU shows up in the diet. For example, if voucher sample X shows that 95% of the reads are OTU y in the data we send you, then when you see OTU y in the diet, you can assume that the species that was provided for sample X could be part of the diet. It can be helpful to compare the species already assigned to an OTU in the closed reference library with the identity of the voucher specimen you provided. They should be closely related.

Can you resolve species within a genus with NGS?

It depends on how different the species are. Sometimes, but generally not. Even if the reference file only matches to one species, that might be because there is only one species of many in the reference database.

How do I manually BLAST a sequence?

The easiest is to go to: http://blast.ncbi.nlm.nih.gov/. Enter in the sequence for an OTU. Results should follow.

Is there any significance to one sample having more total reads than another?

Not really. As long as there is more than ~500 or 1000 reads, the total number of reads conveys little information. Only relative abundance should be compared, not “absolute” abundance.

qPCR Reports

Coming soon...
Jonah Ventures
1600 Range Street Suite 201
Boulder, CO 80301
(785) 409-1655
email: info@jonahventures.com

Jonah Ventures