RADcap: sequence capture of dual-digest RADseq libraries with identifiable duplicates and reduced missing data.

Area of Science:

• Genomics
• Molecular Ecology
• Bioinformatics

Background:

• Genotyping large numbers of individuals and loci is crucial for molecular ecology.
• Existing methods like RADseq and sequence capture face limitations in cost and efficiency.
• High-throughput genotyping requires cost-effective and scalable solutions.

Purpose of the Study:

• To introduce RADcap, a novel method integrating RADseq and sequence capture for efficient and cost-effective genotyping.
• To improve the efficiency of sequencing data usage and sample preparation in molecular ecology.
• To reduce the overall cost per sample for large-scale genotyping projects.

Main Methods:

• Developed RADcap, combining dual-digest RADseq (3RAD) for SNP locus identification with custom sequence capture baits.
• Implemented a new library preparation method to identify and remove PCR duplicates from 3RAD libraries.
• Tested RADcap by genotyping sets of 96-384 Wisteria plants.

Main Results:

• RADcap methodologically reduces PCR duplicates to <5% and allows computational removal.
• Achieved 80-90% on-target reads in 11 of 12 enrichments.
• Provided consistent coverage (≥4×) across >90% of individuals at up to 99.8% of targeted loci.
• Produced high genotype occupancy matrices and demonstrated significant cost reduction compared to current methods.

Conclusions:

• RADcap offers a highly efficient and cost-effective solution for large-scale genotyping in molecular ecology.
• The method ensures consistent data quality and high genotype accuracy across numerous samples and loci.
• RADcap significantly lowers the financial barrier for complex population genetic studies.