Experts in Microsatellite Development

Microsatellites (also known as short tandem repeats) are repetitive DNA elements usually found in non-coding regions of the genome. They have high mutation rates, and therefore are frequently highly polymorphic. Variations in the number of repetitions generate different alleles. This makes them appropriate molecular markers for population genetics and molecular ecology projects.

We develop 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 to 6 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. Our bionformaticians use these reads for primer design. The primers obtained are multiplexed and tested for polymorphism in a number of individuals from different populations.

How we work

Our microsatellite development projects are divided into 3 steps. We can carry out the entire project or only the parts you need.

  • In Step 1 we generate a microsatellite-enriched genomic library. To do this we use high-quality genomic DNA from a number of individuals of your study species. Then, we sequence this library using the Illumina MiSeq platform and obtain thousands of microsatellite-containing reads. Finally, we design 500 primer pairs on the flanking regions of microsatellite motifs. We do this by using the microsatellite-containing reads obtained previously. DNA solutions at a concentration of 10 ng/μL in a minimum volume of 20 μL from 1 or more individuals are required. Alternatively, we can isolate DNA from your samples.
  • In 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. DNA solutions at a concentration of 10 ng/μL in a minimum volume of 100 μL are required per individual. Alternatively, we can isolate DNA from your samples.
  • In 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. The PCR products of these new multiplexes are run in the sequencer and the resulting electropherograms are analysed.

Deliverables include your results, the raw data generated (which will be delivered through our server), and a summary of the methods followed.

Microsatellite markers for a crustacean

A research team from the Universidade da Coruña was interested in developing polymorphic microsatellite markers in a marine crustacean species. They were to be used in a wide population genetic survey.

They gave us ethanol-preserved individuals which our technical team extracted DNA from. A microsatellite-enriched genomic library was constructed and sequenced in a HTS platform‎. Reads were filtered according to quality parameters. Then, those containing appropriate microsatellite motifs were selected for primer design. One hundred primer pairs were multiplexed and tested in 15 individuals from different populations. Our bioinformaticians analysed the electropherograms in order to select those primer pairs amplifying polymorphic regions.

We delivered a comprehensive methodological report, together with all the sequence reads, primers, and labelled probes that had been generated. The researchers were then able to begin the genotyping of crustacean populations.