High-Throughput Sequencing Strategy for Microsatellite Genotyping Using Neotropical Fish as a Model.

Area of Science:

• Conservation Genetics
• Population Genomics
• Molecular Ecology

Background:

• Genetic diversity and population studies are crucial for wildlife management and conservation.
• Traditional microsatellite genotyping is labor-intensive, costly, and requires extensive PCR optimization.
• Efficient methods are needed to analyze multiple genetic loci from numerous samples.

Purpose of the Study:

• To optimize a low-cost genotyping method for simultaneous analysis of multiple microsatellite loci.
• To develop a streamlined workflow for large-scale genetic diversity studies using next-generation sequencing (NGS).
• To adapt bioinformatics pipelines for efficient microsatellite genotyping from NGS data.

Main Methods:

• Designed primers with adapters for combinatorial barcoding amplicon libraries.
• Utilized Illumina MiSeq for sequencing of up to 384 samples.
• Adapted a bioinformatics pipeline for microsatellite genotyping based on read length and sequence content.
• Employed pseudo-multiplex reactions for cost-effective analysis of eight microsatellite loci in fish (Prochilodus costatus).

Main Results:

• A cost-effective methodology was identified using a low-throughput kit for 384 DNA samples.
• An average of 325 reads per individual per locus was observed for eight loci.
• The method allows for testing two to four times more loci per run, depending on PCR quality.
• Bioinformatics analysis effectively eliminated non-specific amplicons, removing the need for exhaustive PCR calibration.

Conclusions:

• A novel, low-cost microsatellite genotyping method using Illumina combinatorial barcoding and NGS was developed.
• This methodology simplifies and accelerates the generation of genotyping data for large-scale conservation genetics studies.
• The approach is promising for efficient wildlife management and biodiversity monitoring.