![]() NGS involves preparation of a full DNA library (no longer one small segment at a time) by amplifying (making many copies) and fragmenting the DNA source of interest (genomic DNA, coding DNA). ![]() Next-generation sequencing (NGS) has revolutionized genetic sequencing capabilities, and moved the fields of science and medicine into the “post-genome” era. This molecular method was revolutionary but very time consuming, labor intensive, and limited to relatively short DNA sequences. Then, by “reading” the terminating base of the synthesized copies from smallest to largest, the sequence of the original single-stranded template is revealed. This generates synthesized fragments of varying length that can be arranged by size, and the reactions containing each terminating base (A, G, C, or T) are kept separated. The original technique of sequencing, called Sanger sequencing, involves synthesizing multiple copies of DNA that is complementary to a single-stranded template of interest using nucleotide-specific chain terminators, or dideoxynucleotide triphosphates (ddATP, ddGTP, ddCTP, ddTTP). This has the potential to uncover pathogenic variants as well as benign variants, and given our limited understanding of how the genome is translated, will potentially identify what are now VUS into a defined clinical phenotype. By listing the full genetic code, variations from an accepted “normal” (reference or consensus sequence) may be discovered. Sequencing determines the complete nucleotide sequence, or specific order of nucleotides in a gene. Robert Resnik MD, in Creasy and Resnik's Maternal-Fetal Medicine: Principles and Practice, 2019 DNA Sequencing
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