Double-digest Restriction site Associated DNA sequencing (ddRAD-Seq)
What Is Double-digest Restriction site Associated DNA sequencing (ddRAD-Seq)?
Double-digest Restriction site Associated DNA sequencing (ddRAD-Seq) is a cost-effective, high-throughput method for sampling consistent subsets of the genome across many individuals—without requiring a reference genome. At the heart of ddRAD-Seq is the choice of two restriction endonucleases with different cutting frequencies:
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Rare Cutter (e.g., PstI, SbfI)
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Recognizes a 6–8 bp motif (e.g. PstI = 5'-CTGCA|G-3'), cutting only every 4–8 kb on average.
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Defines the upstream boundary of your RAD tags.
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Common Cutter (e.g., MspI, MseI)
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Recognizes a 4 bp motif (e.g. MspI = 5'-C|CGG-3'), cutting every ~256 bp on average.
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Adds a second boundary to fragment pools, creating a predictable size distribution.
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Sample-specific barcoded adapters are ligated at these cut sites, libraries are size-selected to a narrow window (e.g., 300–500 bp), and pooled for PCR enrichment and Element sequencing. The result is a reproducible “reduced representation” of the genome, enabling SNP discovery, genotyping, and population genomics in any species.
Advantages of ddRADseq
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Reproducible Loci: Double digest yields consistent fragment sets across samples, avoiding random shearing.
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No Reference Required: De novo locus assembly (e.g., with Stacks, ipyrad) allows SNP calling in non-model organisms.
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Flexible Marker Density: Choose rare/common enzyme pairs and size windows to target hundreds to tens of thousands of loci.
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High Multiplexing: Barcode up to 384 samples per run—perfect for large population studies.
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Cost & Data Efficiency: Sequence ~1–5% of the genome at ~10–20× coverage per locus, reducing reagent and storage costs.
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Population Genomics & Structure: Infer population differentiation, admixture, and demographic history.
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Genetic Mapping & QTL Discovery: Build high-density linkage maps for trait mapping in plants and animals.
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Conservation Genetics: Assess genetic diversity, inbreeding, and connectivity in endangered species.
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Phylogeography & Phylogenetics: Reconstruct evolutionary relationships with genome-wide SNPs.
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Adaptive Genomics: Scan for loci under selection in response to environmental gradients.
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Trait Association Studies: Link SNP markers to phenotypes in breeding and ecological research.
What is ddRADseq Used For?
ddRADseq with AUGenomics
Sample Submission
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Accepted Sample Types: High-quality genomic DNA extracted from fresh, frozen, or dried tissue, blood samples, seeds, leaves, and preserved specimens
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Input Requirement: ≥100 ng genomic DNA recommended for most species; low-input protocols available upon consultation
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Sequencing Recommendations:
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Recommended coverage: typically 10x–100x, depending on species genome complexity
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Suitable for projects requiring high multiplexing capability
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Please refer to our Shipping Guidelines for project-specific guidance.
Turnaround Time
Typical turnaround is 10-14 business days from sample receipt. Expedited options are available depending on project scope and sequencing depth.
Frequently Asked Questions (FAQs)
Q: How do I choose enzyme pairs and size windows?
A: We consult on genome size and GC content to select cutter pairs and size ranges that yield 5–20K loci with even coverage.
Q: Can ddRAD-Seq handle polyploid genomes?
A: Yes—by optimizing allele-calling thresholds and locus clustering parameters, we accurately genotype homeologous loci.
Q: Do you support reference-guided vs. de novo analysis?
A: We offer both: reference alignment where a genome exists, or de novo assembly for non-model species using Stacks or ipyrad.
Got more questions? Contact our team and get a free consultation anytime. info@augenomics.com
