Frequently asked questions• Why does ecogenics recommend enrichment for SSR motifs prior to sequencing?
• What is the turn around time to develop guaranteed polymorphic markers?
• Do you give a discount on the development of microsatellites for multiple species?
• What kind of repeat motifs does ecogenics enrich for?
• What guarantees does ecogenics give?
• Does ecogenics provide guarantees regarding null-alleles?
• How much DNA has to be supplied to begin the development?
• How much DNA has to be supplied for polymorphy testing?
• How do I check DNA quality and quantity?
• Can I send tissue samples from the species of interest instead of extracted DNA?
• Do you also provide DNA extraction?
• What information do I get in the Read&Go brochure?
• Do I get the generated sequence data?
• Are the stock solutions of the primers included in your delivery?
• Will I have the full rights to the markers you developed for me?
• What kind of support do I get after the delivery of the markers?
• How easy will it be to transfer the PCR protocol optimized in your lab to mine?
Why does ecogenics recommend enrichment for SSR motifs prior to sequencing?
The SSR content is species dependent but, usually, less than 2% of the sequence read pool contains a microsatellite insert without enrichment. Our protocols results with 20x enrichment of SSR content and ensures that you receive the best SSR candidates available from the genome of interest. Or in other words, we do not waste sequencing capacity to reads without SSR content.
What is the turn around 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 microsatellites for multiple species?
Due to the massive sequencing power of the used next generation sequencing technology, 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 (with the exception of AT and GC). The customer selects the type of repeat. 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?
Ideally, for the enrichment and the Illumina-based sequencing we need 5 µg of high molecular weight DNA in 50 µl TE or water. This can be a pool of DNA from several individuals.
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.
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.
Can I send tissue samples from the species of interest instead of extracted 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 extraction?
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 optimum 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 (fna 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 support 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 as well as sharing our experience and knowledge on topics such as DNA extraction, non-invasively collected DNA samples, paternity testing, gene-mapping or population genetic 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.