Next-generation sequencing for genetic research is becoming a more and more desirable commodity, which, along with the commercialization of various NGS platforms, is increasingly used by many laboratories worldwide. (Source: Target Enrichment) As can be seen in recent years, computer-aided whole-genome analysis is frequently applied as the basis for cutting-edge discoveries in biomedical research.
Despite the development of first-generation sequencing techniques based on Sanger sequencing, they have become insufficient to achieve some goals. Devices that at that time were considered high-throughput, however, could not match the technology that made its debut in 2005. Genome Analyzer was able to sequence from 84 thousand up to 1 billion base pairs of DNA per cycle.
This technology, which allows for the simultaneous sequencing of vast numbers of short DNA molecules, was a completely new approach in the field and revolutionized the so far known sequencing. Since then, we have seen a considerable increase in the efficiency of NGS equipment while reducing process costs. By 2014, NGS devices could sequence 1.8 terabytes of base pairs in a single device cycle. The process results in an astonishing 1000-fold increase in performance. It sounds particularly impressive when compared to the performance of the devices that sequenced the first human genome in 2001. The project lasted six years and cost $ 3 billion.
Preparation of Libraries
The first step, which is the proper preparation of samples ready for sequencing, is significant for the entire process. Correct sample preparation allows the instrument to operate efficiently and to obtain valuable results. It always consists of preparing so-called nucleic acid libraries (DNA or complementary DNA - cDNA) and duplicating this library. DNA libraries ready for sequencing are constructed by fragmenting DNA molecules (cDNAs) and attaching adapter sequences (synthetic DNA molecules of known sequence) to the ends of the DNA fragments. After the library is built, it is reproduced and ready for sequencing.
Exome sequencing and sequencing of selected regions
Exome sequencing is based on the scrupulous selection of coding sequences (exons) of the human (or other organisms) genome, their enrichment, and sequencing in the NGS technology. Due to the focus on the exome, the efficiency of studying protein-coding regions is very high. The sequencing of selected regions narrows target sequences down to several hundred regions depending on the needs of the study. Exome sequencing and sequencing of selected fragments are mainly used in the research of complex diseases potentially caused by variants (mutations) within the coding regions. The process is of particular importance in the analysis of rare mutations associated with genetic disorders.
Advantages of using NGS in exome/selected fragments sequencing:
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high efficiency and low testing costs
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high exome coverage
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focusing the analysis on the exons only
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facilitating research against existing public genetic databases
Target Enrichment Using Hybridization
In recent years, the method of target enrichment of DNA extract using a hybridization technique has been increasingly adopted. This procedure enables enhancing the share of sequences derived from the species or taxonomic group in the DNA extract even several hundred times. First, DNA is converted into libraries for sequencing. Then they are enriched with the use of appropriate probes. The probes are fragments of DNA
characterized by a high degree of similarity to the studied species or taxonomic group. The probes must be adequately chemically prepared so that they can be easily removed from the reaction later. The probe acts as a "bait" - the desired DNA fragments hybridize to it complementarily. Unhybridized DNA fragments from a different type of organism or group than the test group remain in the solution. In this way, the material to be sequenced is significantly enriched in the DNA fragments of interest. Target enrichment by hybridization is an excellent tool for analyzing particular genetic anomalies in a provided specimen. In the case of environmental studies and analyzes of a larger group of organisms, this method gives complete and more reliable results than the PCR reaction, also in terms of the species composition of a given group.
PCR-Based Target Enrichment
PCR-based, also referred to as amplicon-based, target enrichment methods, utilize primers for amplifying particular areas of interest. Amplification requires a limited volume of DNA data for synchronous targeting of several regions of interest by applying primer pairs for PCR reactions. The advantages of the PCR-based approach are management efficiency, reliable and precise calculation, and its high sensitivity. For analyzing whole-genomes or large genomic regions, amplicon-based target enrichment may be an unfavorable method due to the primers' expenses and the necessity of high DNA input.
The Endless Application of the Next-Generation Sequencing
Next-generation sequencing enables rapid advances in many fields related to biological sciences. Human genome re-sequencing is performed to identify genes and regulatory elements involved in pathological processes. NGS has also provided a tremendous amount of data for comparative biology research by sequencing the entire genomes of many different organisms. NGS also has excellent public health and epidemiological applications through the sequencing of bacterial and viral species to facilitate the identification of new virulence factors.
Additionally, gene expression studies have begun to replace some other technologies to check the level of gene expression. These are only selected applications of NGS technology. They bring undoubted benefits not only to scientists and physicians but also to the entire society.
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