2b-RADseq Service

Overview

CD Genomics provides advanced 2b Restriction Site Associated DNA Sequencing (2b-RADseq) to identify genetic variations, driving discoveries in conservation genetics, population genomics, and evolutionary studies. 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.

To support a broad spectrum of genotyping needs, we provide not only 2b-RAD but also traditional RAD-Seq, ddRAD-Seq, and Genotyping-by-Sequencing (GBS).

What is 2b-RADseq Service

2b-RAD employs type IIB restriction enzymes to cleave genomic DNA at both ends of specific recognition sites. Thus uniform-length fragments (33-36bp) are generated for sequencing. 2b-RAD offers a streamlined and cost-effective alternative to existing reduced-representation genotyping methods. Its key advantage is the ability to screen nearly every restriction site within a genome in parallel. Additionally, 2b-RAD provides flexible control over marker density through the use of selective adaptors, allowing users to optimize the trade-off between genotyping resolution and throughput. This efficiency makes 2b-RAD particularly well-suited for high-throughput genotyping projects operating under constrained sequencing capacity.

Sample Preparation for 2b-RAD Genotyping. (Wang, et al., 2012)

Sequencing Platforms

NextSeq 500

Illumina NovaSeq

PacBio Sequel II

Workflow of 2b-RADseq in Population Genetics

Our 2b-RAD 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 indel identification. Data Format Conversion and Integration	Genetic Map Construction: Identify recombination hotspots. Population Structure Analysis: Identifies genetic subpopulations and quantifies stratification. QTL Mapping: Locates genomic regions.

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, OD260/230>1.5. Clear electrophoresis bands, no smearing.
DNA Quantity: 1–100 ng (conventional samples), trace samples as low as 1 pg (e.g., microorganisms, FFPE tissues).
Sample Handling: Stored at -80℃ for long term reservation.

Demo Results

Distribution of SNPs in chromosomes of cynomolgus macaques.

Heatmap of the differential SNPs between two individuals each within the groups.
(Min et al., J Med Primatol., 2022）

Case Study

Oral microbiota disorder in GC patients revealed by 2b-RAD-M

Journal: Journal of Translational Medicine

Published: 2023

https://doi.org/10.1186/s12967-023-04599-1

Gut microbiota alterations are linked to gastric cancer(GC). However, the relationship between the oral microbiota (especially oral fungi) and GC remains unclear. In this study, researchers applied a technique called "2b-RAD sequencing for Microbiome (2b-RAD-M)" to characterize the oral microbiota in patients with GC. They collected 88 oral samples (44 saliva and 44 tongue coating) from 44 participants. Additionally, 12 gastric tissue samples (6 tumor tissues and 6 adjacent normal tissues) were collected from 6 individuals with GC who underwent surgery. The results showed that oral fungal dysbiosis associated with gastric cancer disrupted the symbiotic bacterial network, likely synergistically promoting the development of gastric cancer. This study is the first to use high-resolution 2b-RAD-M technology to identify oral Malassezia globosa as a potential biomarker candidate associated with gastric cancer. This approach overcomes the limitations of traditional bacterial research and provides new avenues for microecological research.

2b-RAD-M
Bioinfomatics

This study obtained a total of 173,165 microbial genomes (including bacterial, fungal, and archaeal genomes) from the NCBI RefSeq database. Then, built-in Perl scripts were used to sample restriction fragments from microbial genomes by each of 16 type IIB restriction enzymes, which formed an enormous 2b-RAD microbial genome database. Here, they showed the distribution and relative abundance of bacteria in saliva and tongue coating samples from the GC and control groups at the phylum genus, and species levels. The salivary and tongue coating bacteria in the GC and control groups were mainly dominated by Bacteroidota, Proteobacteria, Actinobacteriota, and Firmicutes. 2b-RAD-M achieves precise identification at the species level (e.g., M. globosa vs. S. cerevisiae), whereas traditional 16S rRNA/ITS sequencing only reaches the genus level.

The relative abundance and distribution of salivary bacteria at the phylum, genus, and species levels.

To demonstrate the interconnections between bacteria and fungi with significant differences between the GC and control groups, they constructed chordal diagrams of saliva and tongue coating bacteria versus fungi at the significantly different phylum level. Specifically, saliva and tongue coating Basidiomycota in the GC group were both positively correlated with Bacteroidota and negatively correlated with Ascomycota. Conversely, the phenomenon was reversed in the control group.

Perturbed intrakingdom and interkingdom ecological networks in gastric cancer (GC).
(He, et al. 2023)

FAQs

What is the core advantages of 2b-RAD?

2b-RAD uses uniform tag length (typically 33–36 bp) to eliminate PCR amplification bias and ensures even sequencing depth. It captures all restriction enzyme cutting sites without selective fragment loss. High reproducibility of the technology delivers >95% technical replication rates and >85% marker recovery rates. And it allows compatibility with degraded or trace DNA samples, including challenging sources like formalin-fixed paraffin-embedded (FFPE) tissues or environmental microbiomes with low biomass.

Which research fields benefit most from 2b-RAD applications?

2b-RAD is widely applied in evolutionary biology, ecology, and breeding. It enables population genetics studies to resolve structure and gene flow. It facilitates the development of SNP marker panels for accurate species identification and hybrid detection. Through adaptive evolution analysis, this method identifies signatures of environmental selection by scanning genome-wide selection signals. Additionally, it constructs high-density genetic maps for QTL mapping of key traits. And its modified protocol, 2bRAD-M, resolves species composition in degraded, low-biomass microbiomes with minimal sequencing effort.

How does 2b-RAD mitigate false-positive SNP errors during genotyping?

The protocol minimizes false positives by implementing a bounded SNP model. This model caps allele frequency thresholds to reduce genotyping artifacts. Computational strategies like alignment to reference genomes or algorithms such as the improved Maximum Likelihood (iML) method filter out erroneous signals from repetitive regions or paralogous loci.

References

Wang, S., Meyer, E., et al. (2012). 2b-RAD: a simple and flexible method for genome-wide genotyping. Nature Methods, 9(8), 808-810. http://www.nature.com/doifinder/10.1038/nmeth.2023
Min, F., Xu, F., et al. (2022). Genetic diversity of Chinese laboratory macaques based on 2b-RAD simplified genome sequencing. Journal of Medical Primatology, 51(2), 101–107. https://doi.org/10.1111/jmp.12571
He, S., Sun, Y., et al. (2023). Oral microbiota disorder in GC patients revealed by 2b-RAD-M. Journal of Translational Medicine, 21(1), 246. https://doi.org/10.1186/s12967-023-04599-1

* Designed for biological research and industrial applications, not intended for individual clinical or medical purposes.