ddRAD-Seq Service
Overview
CD Genomics provides advanced Double Digest Restriction Site Associated DNA Sequencing (ddRAD-Seq) to identify genetic variations, driving discoveries in conservation and precise medicine. Our services employ restriction enzymes to digest and target specific genomic regions, enabling cost-effective, high-depth analysis of both genetic and epigenetic variations. Utilizing state-of-the-art sequencing platforms and expert bioinformatics analysis, we deliver high resolution population genomic data tailored to your research needs, from disease studies to population genetics and evolutionary research.
In addition to ddRAD-Seq, we also offer traditional RAD-Seq, 2b-RAD, and Genotyping-by-Sequencing (GBS) services to support a wide range of genotyping applications.
What is ddRAD-Seq Service
ddRAD-Seq employs two different restriction enzymes (REs) to recognize two different restriction sites of genomic DNA. Uniform fragments adjacent to restriction sites are isolated for sequencing. ddRAD-Seq drastically reduces the rate of variant calling and genotyping errors compared with other RAD-Seq approaches. The use of two REs also facilitates adapter design and reduces sequencing costs per individual and per base, thus offering the best multiplexing capability. ddRAD-Seq is suitable for species with low inbreeding, highly heterozygous genotypes and natural populations. So it can be exploited to well distinguish homozygous from heterozygous genotypes, or to approach polyploids, which needs a large depth of coverage.
Diagram of ddRAD-Seq Pipeline. (Andrew, et al., 2016)
Sequencing Platforms
NextSeq 500
Illumina NovaSeq
PacBio Sequel II
Workflow of ddRAD-Seq in Population Genetics
Our ddRAD-Seq service workflow includes sample collection, library preparation, high-throughput sequencing, quality control, and detailed variant analysis to uncover genetic variations and population insights. Customers are encouraged to ensure proper sample handling and share specific research goals for tailored analysis. For any questions about sample requirements, sequencing, or data interpretation, our team is always ready to assist.
Bioinformatics Content
| Basic Analysis | Advanced Analysis |
| Raw Data Quality Control: Per-base sequence quality, GC content distribution and adapter contamination ratio. Sequence Alignment: Retain uniquely mapped reads and mark duplicates. Variant Calling: SNP and indels identification. Data Format Conversion and Integration |
Population Genetics and Phylogeography: Reveals genetic structure, gene flow, and historical demography. Conservation Genetics: Quantifies genetic diversity and inbreeding in endangered species. Phylogenetics and Taxonomy: Resolves species boundaries in taxonomically complex groups. Breeding and Trait Mapping: Facilitates SNP-based fingerprinting and marker-assisted selection. |
Why CD Genomics
- Cutting-edge Sequencing Platforms: Compatible with NGS (Illumina) and third-gen (PacBio/Nanopore) platforms.
- High-throughput, High-quality Sequencing: Deliver premium-quality sequencing data faster, at lower cost, and with unmatched efficiency.
- One-Stop Service: End-to-end analysis from raw data QC, variant calling to population structure analysis, and provide expert data interpretation support.
- Integration of Comprehensive Genomics Analysis: Offer various omics technology services, including advanced and customized genomics analysis solutions.
Sample Requirements
- High DNA Quality: Free from RNA, protein, or exogenous DNA contamination, OD260/280≈1.8-2.0. Clear electrophoresis bands, no smearing.
- DNA Quantity: For initial digestion, minimum input should be 50ng, while optimal input is 100-500ng. For library construction, minimum input should be 20ng, while optimal input is 50-100ng.
- Sample Handling: Stored at -80℃ for long term reservation.
Demo Results
Percentage of total SNP detected by 20X WGS (in orange) and ddRAD-seq (in blue).
Localization of SNPs in (in blue) and out (in light blue) genomic regions theoretically sequenced in ddRADseq.
Comparison of variant calling results between 20X WGS (in orange) and ddRAD-seq (in blue).
( DoubletI et al., PLOS ONE, 2024）
Case Study
Development of Single Nucleotide Polymorphism and Phylogenetic Analysis of Rhododendron Species in Zhejiang Province, China, Using ddRAD-Seq Technology
Journal: Plants
Published:2025
https://doi.org/10.3390/plants14101548
The genus Rhododendron presents significant challenges for systematic classification due to extensive hybridization and adaptive radiation. To resolve phylogenetic relationships among nine ecologically significant Rhododendron species (34 accessions) endemic to Zhejiang Province, China, researchers employed doubledigest restriction site-associated DNA sequencing (ddRAD-seq). Through stringent filtering, they identified 14,048,702 genome-wide single nucleotide polymorphism (SNP). The widespread R. simsii and R. simsii var. putuoense exhibited significant genetic diversity. Whereas the low-altitude widespread R. molle and the endemic R. simiarum exhibited lower genetic diversity. This study provides the first genomic validation of the sibling relationship between R. simsii and its variety, R. simsii var. Putuoense. This study provides both a theoretical framework for SNP-based phylogenetics and critical insights for conserving China' s azalea biodiversity.
- ddRAD-Seq
- Bioinfomatics
ddRAD sequencing was conducted on 34 samples of Rhododendron species, resulting in a total of 39.40 Gb of data, with an average data volume of 1.16 Gb per sample. After filtering, 38.20 Gb of high-quality data was obtained, with an average data volume of 1.12 Gb per sample. A total of 14,048,702 high-quality SNP loci were identified. The number of SNP were counted using a 100 kb sliding window across the populations. Based on the distribution across the 13 chromosomes, the SNP loci showed a relatively even distributed on the chromosomes.
Distribution of SNP loci on thirteen chromosomes of Rhododendron genome.
PCA was conducted to investigate the genetic differentiation among the nine species and one variety of the genus Rhododendron. Based on the positions and mutual distances of the species in the two-dimensional plot, they can be broadly categorized into six groups.
Principle component analysis plot.
Phylogenetic trees based on 14,048,702 SNP loci were constructed using the ML method, revealing that the 34 samples clustered into six well-supported genetic clades. These clustering results are consistent with the findings from the PCA.
Phylogenomic analysis reconstruction .
(Zhu, et al. 2025)
FAQs
ddRAD-Seq employs a dual-restriction-enzyme combination (e.g., EcoRI + MspI) to digest genomic DNA. It eliminates the need for physical shearing and selects target fragment for library construction. Compared to single-enzyme RAD-Seq, this approach offers several critical advantages. This technology provides higher fragment uniformity, lower genotyping error rates and enhanced multiplex sample compatibility.
Optimal restriction enzyme selection requires pairing a rare-cutting endonuclease with a frequent-cutting enzyme to achieve balanced genome coverage. While further optimizing enzyme combinations based on target species-specific genomic features including GC content, repetitive sequences, and methylation sensitivity patterns.
References
- Andrews, K. R., Good, J. M., et al. (2016). Harnessing the power of RADseq for ecological and evolutionary genomics. Nature Reviews Genetics, 17(2), 81–92. https://doi.org/10.1038/nrg.2015.28
- DoubletI, K., Degalez, F., et al. (2024). Variant calling and genotyping accuracy of ddRAD-seq: Comparison with 20X WGS in layers. PLOS ONE, 19(7): e0298565. https://doi.org/10.1371/journal.pone.0298565
- Zhu, H., Li, D. B., et al. (2025). Development of Single Nucleotide Polymorphism and Phylogenetic Analysis of Rhododendron Species in Zhejiang Province, China, Using ddRAD-Seq Technology. Plants, 14, 1548. https://doi.org/10.3390/plants14101548
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