Comparison Of Three RAD-Seq Technologies And How To Choose

With the continual advancement of sequencing technologies, Restriction-site Associated DNA Sequencing (RAD-Seq) has become increasingly instrumental in genomics research. RAD-Seq entails the sequencing of DNA fragments originating from enzyme-digested sites and presents an economically viable approach to generate an abundance of Single Nucleotide Polymorphism (SNP) markers, independent of the availability of a reference genome or considerations of chromosomal ploidy. In order to aid researchers in their selection of the most apt technique tailored to their particular requirements, this article offers a comparative analysis of three widely adopted RAD-Seq methodologies.

Definition of Three RAD-Seq Techniques

Original RAD (Original Restriction-site Associated DNA) : Single enzyme digestion + Mechanical fragmentation for library construction and sequencing.

GBS (Genotyping by Sequencing): Common enzyme single digestion + PCR-based selective amplification of short DNA fragments for library construction and sequencing.

ddRAD (Double-digest Restriction-site-associated DNA): Double enzyme digestion with adapter ligation matching one enzyme + Gel size selection for library construction and sequencing.

Table 1: Comparative Analysis of Three RAD-Seq Techniques

Original RAD GBS ddRAD
Options for tailoring number of loci Change restriction enzyme Change restriction enzyme Change restriction enzyme or size selection window
Number of loci per 1Mb of genome size 30-500 5-40 0.3-200
Length of loci ≤1kb if building contigs; otherwise≤300bp <300bp ≤300bp
Cost per barcoded or indexed sample Low Low Low
Effort per barcoded or indexed sample Medium Low Low
Use of proprietary kit No No No
Identification of PCR duplicates With paired-end sequencing With degenerate barcodes With degenerate barcodes
Specialized equipment needed Sonicator None Pippin Prep
Suitability for large or complex genomes Good Moderate Good
Suitability for de novo locus identification(no reference genome)de novo Good Moderate Moderate
Available from commercial companies Yes Yes Yes

How to choose RAD-Seq Strategy

In accordance with the research objectives and the characteristics of the three simplified genome techniques, four key points should be considered when selecting a strategy.

Reference Genome

Having a reference genome, even if it is of suboptimal quality, proves beneficial for reducing errors in variant detection arising from homologous or repetitive sequences. It also facilitates the detection of InDels and the removal of contaminating sequences. The quality of genome assembly directly influences the outcomes. Furthermore, a reference genome is essential for dependency scans, such as LD analysis and selection analysis. Additionally, a reference genome sequence is required for conducting GWAS (Genome-Wide Association Studies). For species with no reference genome, ddRAD sequencing is recommended.

Sequencing Strategy

(1) For double enzyme digestion, using long reads is not recommended as the insert fragments are short and can lead to adapter contamination. PE sequencing, in contrast, often results in significant overlap.

(2) When insert fragments are longer and the number of reads is the same, long reads can capture more variation information.

(3) With the same data volume, short-read sequencing increases the average sequencing depth for each enzyme-cut tag, enhancing SNP detection accuracy.

(4) For non-reference species, if reads2 from conventional RAD sequencing are not assembled, it will result in a substantial waste of data. In such cases, SE sequencing is recommended.

Recommendation: In the context of species endowed with an accessible reference genome, it is advisable to consider the utilization of conventional RAD sequencing in conjunction with PE151 sequencing. Conversely, for species devoid of a reference genome, the judicious choice would be to employ SE sequencing. GBS and ddRAD methodologies are optimally coupled with PE101 sequencing.

Number of Loci

The number of loci identified in simplified genome techniques is influenced by genome size, the distribution, and quantity of enzyme recognition sites on the genome. Theoretical enzyme-cut fragment counts can be estimated through simulation, depending on the information regarding enzyme recognition sites and genome sequences. For conventional RAD, the aim is to capture all enzyme-cut site-related fragments. However, GBS, which indirectly selects fragments, generally yields a higher number of loci than actual enzyme-cut site-related fragments, which can be adjusted by changing the enzyme type. For ddRAD, the number of loci can be adjusted by both enzyme type and altering the fragment selection range.

Recommendation: For information analysis requiring a high number of markers, conventional RAD sequencing is recommended. For complex genomes and large sample sizes, GBS sequencing is suggested.

PCR Amplification

Introducing Duplicates and Genotyping Errors PCR amplification bias can lead to the detection of heterozygous loci as homozygous or the introduction of PCR amplification errors as true genotypes. It also has a significant impact on information analysis that requires the calculation of sequencing read numbers, such as calculating allele frequencies in pooled samples. For conventional RAD sequencing, PCR duplicates can be mitigated to some extent due to variations in the original library sequence lengths and the fact that both ends are not enzyme recognition sites. However, GBS and ddRAD are less amenable to duplicate removal.

In summary, when formulating a research strategy, it is imperative for researchers to contemplate key factors, including the presence of a reference genome, sequencing approach, loci count, and the potential ramifications of PCR amplification. Each of these considerations holds paramount significance in the discerning selection of the most suitable RAD-Seq technique tailored to the specifics of a given research endeavor.

Simplified genome sequencing has garnered widespread utility within the realm of animal and plant research, serving as a valuable tool for a range of applications including SNP detection, analyses of population evolution, assessments of population structure, evaluations of population diversity, and explorations into the historical dynamics of populations.


  1. Ali OA, O'Rourke SM, Amish SJ, et al. RAD capture (Rapture): flexible and efficient sequence-based genotyping. Genetics, 2016, 202(2): 389-400.
  2. Andrews KR, Good JM, Miller MR, et al. Harnessing the power of RAD-seq for ecological and evolutionary genomics. Nature Reviews Genetics, 2016, 17(2): 81-92.
For Research Use Only. Not for use in diagnostic procedures.
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