Multiplexed microsatellite recovery using massively parallel sequencing.

Area of Science:

• Genomics
• Conservation Biology
• Molecular Ecology

Background:

• Accurate and affordable genotyping is crucial for managing natural populations.
• Traditional microsatellite (simple sequence repeat - SSR) analysis is a widely used, cost-effective genotyping method.
• Advancements in sequencing technology enable new approaches for marker discovery.

Purpose of the Study:

• To evaluate the efficacy of using multiplexed massively parallel sequencing (MPS) for identifying microsatellite markers.
• To assess the cost-effectiveness and conversion rates of MPS for microsatellite genotyping.
• To determine the feasibility of applying this method for large-scale conservation initiatives.

Main Methods:

• Preparation of seven multiplexed, barcoded, microsatellite-enriched genomic libraries from diverse taxa (conifers and birds).
• Sequencing of libraries using Illumina Genome Analyzer with paired-end 80-bp reads.
• Analysis of millions of sequences to identify microsatellite-containing reads and assess marker conversion rates.

Main Results:

• Screened 6.1 million sequences, identifying 356,958 unique microsatellite-containing microreads.
• Achieved favorable conversion rates from raw sequences to polymorphic markers compared to existing methods (Sanger, 454).
• Reduced per-species preparation and sequencing costs to under $400 USD through multiplexed MPS.

Conclusions:

• Multiplexed MPS provides an accurate, inexpensive, and high-throughput method for microsatellite marker discovery.
• This approach significantly lowers genotyping costs, making it viable for conserving numerous threatened and endangered species.
• The method holds substantial promise for advancing population genetics and conservation management.