Microsatellite marker development

Microsatellite Marker Development

Microsatellites (short tandem repeats like GAGAGAGA) are repetitive DNA elements that tend to occur in non-coding regions of the genome. They have high mutation rates, and therefore are frequently highly polymorphic. In some microsatellites, the repeated unit (e.g. GA) may occur four times, in others it may be six or ten or thirty. Variations in the number of repetitions generate different alleles. This makes them appropriate molecular markers for population genetics and molecular ecology projects. StarSEQ offers a next generation sequencing based method to develop microsatellite markers for any species within a short time. Our well proven service is customizable, scalable and divided into 4 phases:

Phase 1: gDNA preparation service (optional)

• Genomic DNA preparation from frozen or stabilized reagent preserved tissues
• Grinding with ball mill homogenizer
• Contamination free with lysis beads in screw cap tubes
• Quantification with fluorescence assay
• Ready for gDNA library preparation and other downstream applications

Phase 2: Identification of microsatellite markers

• Quantification and QC of genomic DNA
• DNA sequencing library suitable for microsatellite development from genomic DNA
• Size selection optimized for maximization of short overlaps from paired-end reads
• Sequencing of genomic DNA with Illumina™ MiSeq, 1 full Flow Cell, 2 x 300 nt paired end, up to 50 million reads and up to 15 Gb
• Merging of overlapping paired-end reads
• Bioinformatic screening for microsatellite containing sequences of Illumina™ reads
• Selection of 96 microsatellite containing sequences suitable for primer development and sufficient for development of 10 marker PCR assays
• Design of primer pairs for selected microsatellite loci

Phase 3: Development and analysis of PCR assays for microsatellite loci (optional)

• Synthesis of unlabeled primers for selected microsatellite loci
• Testing of PCR assays on 16 independent individuals of the same species
• Sanger Sequencing of 4 PCR products per assay to confirm polymorphic alleles
• Assay report

Phase 4: Fragment length analysis (optional, 96 well format only)

• Synthesis of labeled and unlabeled primer pairs (dye set DS-33) for selected microsatellite loci
• Accomplishment of PCR with labeled primer and clean-up
• Dilution of PCR, adding of LIZ 500 Standard and HiDi-Formamide
• Fragment length analysis with ABI 3730 and providing fluorescent trace files (.fsa)
• Optional: analysis of fragments from individuals usinig Geneious or GeneMapper software