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CD Genomics is now providing 2b-RAD sequencing, which is a restriction-site associated DNA (RAD) sequencing and a novel reduced representative whole genome sequencing method to enable genome-wide genotyping in a cost effective way.


2b - RAD is based on type IIB restriction enzymes (RE), which share the feature of cutting the genomic DNA at both sides of the recognition site at a fixed distance, resulting in protruding noncohesive ends. The result is short genomic DNA fragments of identical size at each IIB RE site in the genome. The technology overcomes some shortcomings of RAD-Seq and GBS because of its use of spe­cial restriction enzymes that can exclusively produce isolength tags (32–36 bp). Sequencing of these short and uniform tags can bring many benefits, including targeting of all restriction sites; even sequencing depth across sites; high reproducibility for quantitative measurement; low sequencing cost per tag; and low sensitivity to DNA degradation. To benefit from the gradually increased sequencing capacity of next-generation sequencing (NGS) platforms, we also combine an advanced protocol that allows the preparation of five concatenated isoRARAD tags for Illumina paired-end (PE) 150 bp sequencing, which provides researchers more power and flexibility in devising effective library configurations to meet specific research purposes (Wang et al., 2016).


The library construction for 2b-RAD is simple, which consists of four major stages (digestion, ligation, amplification and barcoding). Following DNA digestion by BsaXI, adaptors are ligated to the fragments through cohesive-end, and specific barcodes are incorporated into each sample through PCR amplification using degenerated linkers. Then the single-tag constructs are produced using modified adaptors and biotin-labeled primers, which can be further digested by SapI to generate distinct cohesive ends and then ligated in a predefined order to produce five concatenated tags. Samples are then pooled and sequenced using Illumina technology. The workflow is illustrated in Figure 1 (Wang et al., 2012).

Schematic overview of the 2b-RAD procedure Figure 1. Schematic overview of the 2b-RAD procedure.

Technique Data

  1. Genome label average density: 2 kb
  2. The average depth of label ≥ 20X
  3. Classification accuracy ≥ 95%

Data Analysis:

  1. Data quality control
  2. High quality label acquiring
  3. Label clustering
  4. SNP classification based on iML
  5. Mark separation analysis
  6. Bin Map construction


  1. Bin Map construction and QTL location
  2. Population genetic study
  3. Population evolution analysis
  4. Genome-wide association study
  5. Genome phylogeny
  6. Genomic selection
  7. Linkage and association mapping
  8. Discrimination of microbial strains
  9. Detection of somatic mutations

Sample Requirements

  1. DNA amount: ≥ 1 µg; DNA concentration: ≥ 200 ng/µl; OD 260/280: 1.8~2.0
  2. Species: Diploid organisms with or without reference genome

Key Features and Advantages

  • Strong flexibility. Tag number is under control
  • Consistent label length. Benefit from consistent PCR amplification efficiency.
  • Position specificity in labels. Does not depend on the reference sequence and assembly result
  • Extremely even read productivity across samples
  • Tunable genome representation
  • Detection of strand bias
  • Allowing for extremely low DNA input by using selective adaptors
  • Comprehensive data analysis. Based on mixed poisson distribution model of de novo SNP classification (iML), which can remove the distraction of repetitive sequence effectively. Classification accuracy is better than existing algorithms, with a false positive rate is lower 20%.


CD Genomics is proud to offer advanced 2b-RAD sequencing technology, it harbors numerous advantages over original RADseq and GBS, the comparison between different reduced representation whole genome sequencing method is outlined in Table 1.

Table 1. Technical comparison between 2b-RAD and other NGS-based reduced representation genotyping methods.

Technology RAD-seq, ddRAD GBS 2b-RAD
Library Construction Complex, including sonication, fragment selection and multiple steps of DNA purification No sonication and fragment selection steps, end-repair Streamlined, no sonication and fragment selection steps, end-repair
Fragment Size Non-uniform Non-uniform Uniform
Tag Density Adjustment Difficult Possible but not tested Flexible
Tag Sequencing Depth Highly divergent depth between different tags Highly divergent depth between different tags Uniform sequencing depth between tags
Data Analysis False positive caused by repetitive sequences False positive caused by repetitive sequences Remove the distraction of repetitive sequence by using iML

Our assayed organisms are also highly diverse, ranging from bacteria to plants to animals, and from small to large and complex genomes.


  1. Wang S. et al. Serial sequencing of isolength RAD tags for cost-efficient genome-wide profiling of genetic and epigenetic variations. Nature Protocols. 2016, 11(11): 2189-2220.
  2. Wang S. et al. 2b-RAD: a simple and flexible method for genome-wide genotyping. Nature Methods. 2012, 9(8): 808-810.
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