Pros and Consįast and cost-effective for low target numberEstablished workflowSimple data analysisLonger reads (500-700 bps) Therefore, it is relatively easier and faster, especially for gene sequences with repeats, which remains a big challenge for NGS platforms that require linking short sequence reads together (e.g., Illumina NGS). Partly, this is because Sanger sequencing can read up to 500-700 bps per reaction without complicated data analysis. Yet, Sanger sequencing remains a preferred method in the molecular biology laboratory for sequence verification of genes and plasmids. Discovery applications requiring sequencing of a large number of genes (e.g., over 100 gene targets), or high sensitivity and therefore greater reading depth, as needed for complex samples (e.g., tumor tissue), would benefit from next-generation approaches both in cost and turn-around time. Therefore, when it comes to choosing between next-generation and Sanger, both highly accurate DNA sequencing approaches, sample volume remains a primary differentiating factor (Slatko et al. The Sanger method allows the sequencing of one DNA fragment at a time. However, more studies are emerging supporting the accuracy of NGS methods for variant identification and questioning the redundant use of a more time-consuming and costly process for validation. In fact, most clinical laboratories rely on Sanger sequencing to validate gene variants (e.g., single-nucleotide variants and insertion/deletions) identified first through NGS. With over 99% accuracy, the Sanger sequencing method remains the “gold standard” for basic and clinical research applications. Retrieved from Choosing between Sanger sequencing and NGS Reprinted from “Sanger Sequencing”, by (2021). 5- A chromatogram is generated based on peak fluorescence detected, corresponding to fluorophore-labeled ddNTPs incorporated at each position of the DNA sequence. 4- Labeled DNA fragments are separated based on molecular mass by capillary gel electrophoresis, and their associated fluorescence is detected by a Charge-Coupled Device (CCD). 3- DNA fragments of various lengths and labeled with different fluorophores are generated. 2- Binding of a fluorophore-labeled ddNTP leads to termination of chain extension. Polymerase catalyzes the DNA chain extension by random incorporation of available nucleotides. Sanger sequencing method. 1- Deoxynucleotides (dNTPs) and a low concentration of fluorophore-labeled dideoxynucleotides (ddNTPs) are supplied together with DNA template and primers. Reiteration of primer annealing and DNA extension cycles results in fragments with a fluorophore-labeled nucleotide at each position, thereby identifying every nucleotide in the DNA template (Crossley et al. Because ddNTPs lack a hydroxyl group needed for nucleotide binding, the addition of ddNTPS by the DNA polymerase during chain extension terminates the DNA strand elongation. Sanger sequencing, or the enzymatic chain termination method, is based on the use of fluorophore-labeled dideoxynucleotides (ddNTPs) in combination with regular deoxynucleotides (dNTPs). Nevertheless, Sanger sequencing remains a method of choice, especially for the analysis of low-volume DNA sequences, as it provides high-quality DNA sequencing data for regions of up to 1,000 bases. Next-generation technologies have undoubtedly reduced DNA sequencing costs and expedited high-quality genome sequencing. Nevertheless, NGS or second-generation technologies share their reliance on massively parallel processing and the potential to sequence thousands to millions of DNA strands simultaneously. Over the years, different NGS methods and platforms have been developed based on unique chemistries and detection methods (e.g., Pyrosequencing, Reversible-dye terminator, and Proton detection) (Garrido-Cardenas et al. Next-Generation sequencing (NGS) technologies evolved from the need to sequence larger volumes of genetic material faster and at a lower cost. Ultimately, the Sanger chain termination or dideoxy method, also first reported in 1977, provided the foundation for fast-paced growth in DNA sequencing technologies and enabled sequencing the human genome for the first time in 2001 ( bSanger et al. DNA sequencing technology rapidly advanced from its inception in the 1970s with the work of Frederick Sanger, who sequenced the first full genome based on the “plus and minus method” ( aSanger et al. DNA sequencing is the process of identifying the exact order of nucleotide bases (i.e., Adenine, Cytosine, Guanine, and Thymine) encoding specific genomic information.
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