Frequently Asked Questions
General
How long do you store my samples and data?
We will store raw data files and your samples and project specific chemicals such as PCR or sequencing primers after completion of the project for 6 months. Afterwards raw data files will be deleted and samples and chemicals properly disposed of. On request, we can send the samples/chemicals to your lab. Please note that we will charge an additional fee for sample shipment.
How do I check DNA quality and quantity?
For testing DNA quality, please run about 100 ng of your extracted DNA on a 1.5% Agarose gel. Non-degraded DNA will run as a distinct homogenous band of high molecular weight. Please make sure that your DNA is not degraded and free of RNA (no low molecular smears visible). While many labs use absorbance readings at 260 nm for DNA quantification, there are serious limitations on this technique. Contamination from proteins, carbohydrates, ssDNA, RNA, and free nucleotides contribute significantly to the reading, often making the results a "best guess". We therefore recommend to quantify DNA by using fluorescent dyes such as Hoechst 33256, PicoGreen or similar compounds. However, ecogenics will always check DNA quality and quantity of your samples before starting the development.
Development of SSR's
What is the turnaround time to develop guaranteed polymorphic markers?
Typically, it takes 8 to 10 weeks from the day we receive the DNA through to the test for polymorphism. If needed, we can achieve project completion even faster.
Do you give a discount on the development of SSRs for multiple species?
Due to the massive sequencing power of the next generation sequencing technology that we use, we can analyze several species in parallel, yielding significantly lower prices for the simultaneous development of microsatellite markers for multiple species! Please contact us for a custom-made offer.
What kind of repeat motifs does ecogenics enrich for?
We enrich for any kind of di-, tri-, and tetranucleotide repeat motif. The choice will largely depend on the research question, the target organism and the screening technology used afterwards. We are happy to provide you with our expert opinion about the best choice for your target species.
What guarantees does ecogenics provide?
For our guaranteed polymorphic markers, we guarantee at least 4 alleles in 15 unrelated individuals. If the developed loci are less polymorphic, we will deliver more loci instead, to reach the guaranteed total number of alleles.
Does ecogenics provide guarantees regarding null-alleles?
ecogenics cannot provide specific guarantees regarding null-alleles because their occurrence depends on the genetic characteristics of the species and not on our development abilities. We routinely test our markers on 15 individuals and usually discard markers that do not amplify in all 15 individuals. However, if the species is prone to null-alleles, and many of the tested markers are affected, we cannot uphold this strategy and will also have to deliver markers that do not amplify in all individuals.
How much DNA has to be supplied to begin the development?
Per species we ideally need > 50 µl clean, non-degraded genomic DNA with a concentration of >20 ng/µl (= 1 µg). gDNA can be sent in screw cap tubes with AE, 10 mM Tris-HCL buffer (pH 7.5 - 8.5) or water.
How much DNA has to be supplied for polymorphy testing?
In order to test polymorphy of the markers, we need DNA of 15 unrelated individuals. Please provide 1 µg DNA of each individual in separate tubes. It is your responsibility to ensure that the 15 sampled individuals represent the species genetically.
Can I send tissue samples from the species of interest instead of isolated DNA?
You are welcome to send us tissue samples (skin biopsy from tissue punches, blood, hair, faeces, bones, feathers, swabs etc.) from your organism of interest. Please contact us to discuss suitable measures to avoid DNA degradation. Be aware that DNA used for sequencing must be free of contaminating DNA from other organisms. Samples such as faeces or swabs are prone to be contaminated with foreign DNA, hence, you risk developing markers for the contaminating organisms. However, the DNA samples used for polymorphy tests are less critical towards contamination with foreign DNA. This type of samples must be free from contaminations with DNA from another individual of the same species.
Do you also provide DNA isolation?
Yes, ecogenics provides DNA extraction services for any kind of organism. We have vast experience in extracting DNA even from very difficult sources, such as certain plant and fungi species, snails, feathers, hair, horn, faeces etc. In the context of microsatellite development projects, we provide DNA extraction services free of charge.
What information do I get in the Read&Go brochure?
The Read&Go brochure is a great source for all the information needed to use and publish your newly developed markers. It contains a primer certificate for each locus with all relevant information, including the sequence of the microsatellite-containing region and electropherograms representing the alleles found in 15 unrelated individuals and the used PCR conditions. If ordered, it contains the information for multiplex PCR. In addition, it covers all details needed to publish the markers. The Read&Go brochure also gives you many useful tips on how to get the most out of your markers, such as specific suggestions on how to approach a multiplex PCR design yourself.
Do I get the generated sequence data?
You will receive all generated sequence data (fastq, fasta and qual files) from your project as well as separate text files of all sequences containing microsatellite motifs.
Are the stock solutions of the primers included in your delivery?
Yes, included in our delivery you receive the 100 µM stock solutions of the primers. Also included will be a primer certificate for each locus with all relevant information. The latter information is compiled in your Read&Go brochure.
Will I have the full rights to the markers you developed for me?
Yes, the full rights to the delivered markers, sequences and primers belong to you. ecogenics will not give out any information to third parties without asking for your consent. In addition, it is up to you to decide whether or not you would like to offer ecogenics a co-authorship on a primer note or publication.
What kind of supports do I get after the delivery of the markers?
Your satisfaction is our goal! We will gladly support and advise you if any questions arise from the use or application of the delivered markers. We will further be of assistance if questions arise in data interpretation and analysis.
How easy will it be to transfer the PCR protocol optimized in your lab to mine?
It will be straightforward! The PCR conditions for our markers are very robust. In order to enable multiplex PCR, we use whenever possible a standard PCR protocol for amplifying all microsatellites. However, in some cases we must adapt the thermocycling program to ensure proper amplification. Of course, you will get all this information in your Read&Go brochure.
Genotyping-by-Sequencing
Do you conduct a quality control before library preparation?
Yes, we conduct a thorough entry quality control of your samples. Nevertheless, please ensure that you do your own quality control before you ship the samples in order to avoid delays or other pitfalls in processing.
How do sequencing depth, read length and replicates impact my project?
To put it simply:
- Sequencing depth means sensitivity
- Read length means specificity
- Replicates mean confidence
