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The Charm of High-Throughput Sequencing: How Can RAD Markers Change the Game Rules of Genetics?
With the rapid development of genomics, new tools and technologies for genetic research are constantly being introduced, and the emergence of restriction site-associated DNA (RAD) markers has undoubtedly brought revolutionary changes to the field. This new type of genetic marker can not only facilitate association mapping, QTL mapping, population genetics and other studies, but also shows strong potential in ecological genetics and evolutionary genetics.
The charm of RAD markers lies in their ability to quickly and efficiently scan polymorphisms in the genome, providing an unprecedented means for genetic research.
When performing RAD labeling, the core process is to isolate RAD tags, which are DNA sequences near each specific restriction site of a restriction enzyme in the genome. The advantage of this approach is that researchers can more accurately identify and genotype, especially single nucleotide polymorphisms (SNPs). Although the emergence of high-throughput sequencing technology has brought new challenges, it has also made the application of RAD markers a possible and cost-effective option.
The separation process of RAD tags
Isolation of RAD tags requires digestion of DNA with specific restriction enzymes, followed by ligation of biotin-labeled adapters to hangs. This process randomly cuts DNA into fragments smaller than the distance between restriction sites, and then uses streptomycin beads to isolate the biotin-laden fragments. Such operations were originally prepared for microarray analysis, but with the advancement of technology, high-throughput sequencing is now commonly used to perform this process, which greatly improves the processing power and accuracy of data.
New tag separation procedures are an important part of the high-throughput sequencing process, making genome analysis more efficient.
Detection and genotyping of RAD markers
After isolating back the RAD tags, the next step is to use these tags to identify polymorphisms, such as SNPs, in the DNA sequence. It is worth noting that previous microarray methods have certain limitations in the identification of RAD markers due to their low sensitivity and inability to effectively detect all polymorphic changes. On the other hand, with the promotion of high-throughput sequencing technology, higher gene marker density can be achieved, which allows researchers to deeply explore the diversity of genomes and accelerate their understanding of the relationships between species.
Historical background and development
The first application of RAD markers dates back to 2006 and was developed by Eric Johnson and William Cresko at the University of Oregon. Initially, they used RAD markers to identify recombination breakpoints in Drosophila and detected QTLs in three-spined squid. Over time, RAD labeling technologies have evolved, becoming more powerful and diverse, such as the double-digested RADseq (ddRADseq) technology in 2012, which enables cost-effective scanning, especially in whole-genome selection and population adaptation. Excellent performance.
Innovative approach: hyRAD
In 2016, researchers proposed a new method called hybrid capture RAD (hyRAD), which uses biotin-labeled RAD fragments as probes to effectively capture homologous fragments from genomic libraries, so that even in Analysis of highly degraded DNA samples can also be performed. This approach not only reduces reliance on restriction sites, but also allows researchers to explore the diversity of the genome more broadly.
The emergence of hyRAD has opened up a new research space in related research fields such as paleontology and natural history, providing us with more possibilities for understanding the evolutionary background of species.
The introduction of high-throughput sequencing technology makes the application of RAD markers no longer limited to research laboratories, but can be more widely used in ecosystem research. Its advantage is that it can analyze multiple species at once and effectively connect the relationship between genomic data and biological phenomena. With the further development of these technologies, what kind of breakthroughs and innovations will genetic research bring in the future?
