![]() ![]() ![]() As the molecules migrate in the presence of the electric field, they flow past a detector that registers the fluorescence intensity and color, yielding a series of peaks that can be mapped directly to a DNA sequence. In Sanger sequencing, an ensemble of DNA molecules-all originating from the same position on the template but having different size due to termination at different positions-are arranged in an electric field, which separates them by size because DNA is negatively charged. The fundamental challenge for the sequencer, then, is to organize molecules such that their fluorescence signal is interpretable. In both sequencing techniques, when polymerase incorporates a modified base into the copied strand, extension of the new strand stops, and, critically, this newly terminated strand is uniquely colored to reflect its most recently added base. In Sanger sequencing, only a small percentage of bases are modified, whereas in NGS, all available bases are modified. There are four different colors of modified bases for A, T, G, and C (Fig. The key innovation that transforms DNA replication into the DNA-sequencing strategy at the core of both Sanger and NGS is the use of unextendable, fluorescently labeled modified bases. Extension, arrangement, and detection are shared steps in both protocols but occur in different order, with NGS alone having a restoration step that converts bases to the undyed and extendable form In both methodologies, a polymerase copies template molecules by incorporating nucleotides from a pool, that is, either partially (Sanger) or entirely (NGS) composed of dyed and unextendable bases. ![]() NGS is a slightly modified, digital, and vastly scaled-up implementation of Sanger sequencing. In a conceptually simplified form, DNA replication requires only three types of molecules: a template strand, free bases, and a polymerase enzyme that links the free bases together one-at-a-time into a new strand complementary to the template (Fig. However, because the predominant technique used in genetic medicine today is the sequencing-by-synthesis approach employed by Illumina devices, here we use the term NGS to refer specifically to Illumina-style sequencing.Įven though NGS is largely displacing Sanger sequencing in molecular diagnostics, the two technologies share a common origin that dates back millions of years: both repurpose the DNA replication machinery that copies DNA during every cell division. These innovations include sequencing-by-synthesis, sequencing-by-ligation, ion semiconductor sequencing, and others. The term “NGS” does not denote a single technique rather, it refers to a diverse collection of post-Sanger sequencing technologies developed in the last decade. Here we discuss how NGS is exquisitely capable of revealing both types of variants in patients’ genomes. Such genomic differences-called “variants”-fall into two classes: (1) changes to the DNA sequence, e.g., the single-nucleotide polymorphisms (“SNPs”) and short insertions/deletions (“indels”) in the CFTR gene that can cause cystic fibrosis, and (2) large deletions/duplications (“del/dups”, a.k.a., “copy-number variations” or “CNVs”), e.g., the whole-gene deletions of HBA1 and HBA2 that largely determine the presence and severity of alpha-thalassemia. The clinical utility of an NGS-based test stems from its ability to confidently identify the differences between a patient’s genome and the reference genome. These advances have allowed NGS-based tests to enter the clinic, where they are an exponentially growing presence in carrier screening, testing for fetal aneuploidies, detecting the presence of rare diseases, and assessing both the risk and existence of cancer. Remarkably, not even 15 years after decoding the first human genome, NGS techniques now enable the sequencing of an entire human genome in a single day for around $1000. ![]() Therefore, in order for the theoretical promise of personalized genomic medicine to become a clinical reality, a quantum leap in sequencing technology was required. Unfortunately, by the end of the Human Genome Project in 2002, this mature sequencing technique was already operating at nearly peak efficiency, making it totally unsuitable for scaling up to the task of sequencing millions of patients’ genomes quickly and affordably. The effort involved hundreds of researchers around the world and was a tour de force of the “first-generation” Sanger sequencing technology developed in the 1970s. Assembly of the first human genome sequence consumed 12 years and cost nearly $3 billion. ![]()
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