Experts in microsatellite development
Microsatellite markers for your study species
At AllGenetics, we use high-throughput sequencing to obtain primer pairs which amplify polymorphic microsatellite loci in your study species. Genomic DNA is used to generate genomic libraries. We usually enrich these libraries with 4 different microsatellite motifs. However, we can customise the number of motifs to your needs. We obtain thousands of microsatellite-containing reads by using high-throughput sequencing. These reads are used for primer design. The primers obtained are multiplexed and tested for polymorphism in a number of individuals from different populations.
What you receive
Step 1: a summary of the methods followed, ~3 million MiSeq PE300 reads, and a list of 500 non-tested primer pairs which amplify microsatellite loci in your study species.
Steps 1+2: everything in step 1 + a list of the primer pairs biologically tested, the primers and fluorescent probes, the ready-to-use PCR protocol, and the multiplex setup.
Steps 1+2+3: everything in step 1 and step 2 + the optimised multiplex setup.
If you require additional analyses, please let us know and we will do our best to meet your needs.
What we need
We can start working by using either your tissue samples, or already extracted DNA.
If you are sending tissue samples, please make sure that they have been properly preserved for DNA isolation (ethanol, silica gel, frozen, or another suitable preservation method). If required, we can provide sampling kits and sample collection guidelines to ensure that your samples arrive at our lab in optimal conditions.
If your DNA has already been extracted, we ideally require a minimum volumen of 50 μL of DNA in TE buffer or water at a minimum concentration of 10 ng/μL, and 260/280 and 260/230 absorbance ratios higher than 1.8.
Please note that, depending on the specifics of your project, these requirements may vary. Please contact us for the specific requirements of your project.
How we work
Step 1: we generate a microsatellite-enriched genomic library. To do so we use high-quality genomic DNA from 1 individual of your study species. Then, we sequence this library using the Illumina MiSeq platform and obtain thousands of microsatellite-containing reads. Finally, based on the microsatellite-containing reads obtained previously, we design 500 primer pairs on the flanking regions of microsatellite motifs.
Step 2: primer pairs will be multiplexed in sets of 3, based on their specific features. These primers will be tested in a number of individuals. You decide how many primer pairs you would like us to test and the number of individuals you would like us to genotype. Even though the number of primer pairs amplifying polymorphic loci is extremely dependent on the biological features of the species under study, as a general rule we suggest to test 4 times as many primer pairs as polymorphic loci you need for your project.
Step 3: we select only those primer pairs which amplified polymorphic loci in Step 2. Then, we rearrange the multiplexes in order to optimise them and test them in 3 individuals. The PCR products of these new multiplexes are run in the sequencer and the resulting electropherograms are analysed.
For your convenience, we can carry out the entire project, step 1 alone, or steps 1+2. Please contact us for further information and pricing.