Method Category: Genome > Rearrangements and Markers

Description: Double digest restriction-site associated DNA (ddRADseq), also called ddRAD, is a variation on the RAD sequencing protocol, which is used for SNP discovery and genotyping. , In this variation, the fragment shearing is replaced with a second restriction digestion to improve the tunability and accuracy of the size-selection step. The protocol also includes a second index to allow combinatorial indexing. Several RAD variations 2b-RAD, SLAF-seq, and hyRAD have been developed to address specific applications and there are multiple software packages available to analyze RAD data.

In this method, genomic DNA is first digested with a restriction enzyme and a barcoded P1 adaptor ligated to the fragments. The adaptor-ligated fragments from different samples are combined, if samples are multiplexed, and the DNA digested by a second restriction enzyme. The fragments are size-selected and purified. The P2 primers are then ligated and the fragments amplified.

  • No reference genome is required1
  • Relatively inexpensive, compared to whole-genome sequencing
  • The degree of genome coverage can be adjusted by selecting various restriction enzymes
  • There can be gaps in the genome coverage
  • Requires high-quality DNA (See hyRAD for low quality DNA)

Related Publications

  1. DaCosta J. M. and Sorenson M. D. (2016) ddRAD-seq phylogenetics based on nucleotide, indel, and presence-absence polymorphisms: Analyses of two avian genera with contrasting histories. Mol Phylogenet Evol 94: 122-135
  2. Lal M. M., Southgate P. C., Jerry D. R. and Zenger K. R. (2015) Fishing for divergence in a sea of connectivity: The utility of ddRADseq genotyping in a marine invertebrate, the black-lip pearl oyster Pinctada margaritifera. Marine Genomics
  3. Brown J. K., Taggart J. B., Bekaert M., Wehner S., Palaiokostas C., et al. (2016) Mapping the sex determination locus in the hapuku (Polyprion oxygeneios) using ddRAD sequencing. BMC Genomics 17: 448
  4. Clark L. V. and Sacks E. J. (2016) TagDigger: user-friendly extraction of read counts from GBS and RAD-seq data. Source Code Biol Med 11: 11, Hou Y., Nowak M. D., Mirre V., Bjora C. S., Brochmann C., et al. (2015) RAD-seq data point to a northern origin of the arctic-alpine genus Cassiope (Ericaceae). Mol Phylogenet Evol 95: 152-160
  5. Shirasawa K., Hirakawa H. and Isobe S. (2016) Analytical workflow of double-digest restriction site-associated DNA sequencing based on empirical and in silico optimization in tomato. DNA Res 23: 145-153
  6. Wu Z., Wang B., Chen X., Wu J., King G. J., et al. (2016) Evaluation of Linkage Disequilibrium Pattern and Association Study on Seed Oil Content in Brassica napus Using ddRAD Sequencing. PLoS One 11: e0146383
  7. Yang G. Q., Chen Y. M., Wang J. P., Guo C., Zhao L., et al. (2016) Development of a universal and simplified ddRAD library preparation approach for SNP discovery and genotyping in angiosperm plants. Plant Methods 12: 39
  8. Cai G., Yang Q., Yi B., Fan C., Zhang C., et al. (2015) A bi-filtering method for processing single nucleotide polymorphism array data improves the quality of genetic map and accuracy of quantitative trait locus mapping in doubled haploid populations of polyploid Brassica napus. BMC Genomics 16: 409
  9. Davik J., Sargent D. J., Brurberg M. B., Lien S., Kent M., et al. (2015) A ddRAD Based Linkage Map of the Cultivated Strawberry, Fragaria xananassa. PLoS One 10: e0137746
  10. Leache A. D., Banbury B. L., Felsenstein J., de Oca A. N. and Stamatakis A. (2015) Short Tree, Long Tree, Right Tree, Wrong Tree: New Acquisition Bias Corrections for Inferring SNP Phylogenies. Syst Biol 64: 1032-1047
  11. Leache A. D., Chavez A. S., Jones L. N., Grummer J. A., Gottscho A. D., et al. (2015) Phylogenomics of phrynosomatid lizards: conflicting signals from sequence capture versus restriction site associated DNA sequencing. Genome Biol Evol 7: 706-719
  12. Meik J. M., Streicher J. W., Lawing A. M., Flores-Villela O. and Fujita M. K. (2015) Limitations of Climatic Data for Inferring Species Boundaries: Insights from Speckled Rattlesnakes. PLoS One 10: e0131435