Experts in microsatellite development
Microsatellite markers for your species wih our microsatellite development service
AllGenetics' microsatellite development service uses high-throughput sequencing to obtain primer pairs which amplify polymorphic microsatellite loci in your study species. The primers obtained are multiplexed and tested for polymorphism in a number of individuals from different populations.
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 - 5, 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.
What you receive
A report with a summary of the methods followed.
The raw data files, which will be delivered through our server.
Step 1: ~3 million Illumina 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.
At no additional cost, our project specialists will be available to you after project delivery and during the editorial process of your scientific articles. We offer up to five 60-min sessions per project, depending on the project size.