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How Does NGS Compare to First Generation Sequencing?
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This form of sequencing became the main tool for the completion of the human genome project. AB370 could detect 96 bases at one time, 500K bases per day, and had a read length of 600 bases by using a parallel analysis and high throughput setup. The biggest difference here was speed and cost. In this way, the genome is carefully read. As the fragments are pulled toward the positive electrode of a capillary (see image below), they pass a laser beam that triggers a flash of light from the fluorochrome attached to the ddNTP that is characteristic of the base type (for example, green for A, yellow for T, blue for G, red for C). Take the above example of ddNTP incorporation and imagine that the bold letters are ddNTPs with a fluorochrome attached. The more negative the charge, the longer the fragment. Size is measured by the PCR product’s overall negative charge. By the Sanger sequencing method, PCR products of various lengths are created, and then separated according to their size. The machine relied on a method called capillary electrophoresis that used Sanger’s chain terminating method without needing a gel.įluorescently labelled chain-terminating ddNTPs are added to the PCR reaction mix. In 1987, Applied Biosystems became the first to introduce an automatic sequencing machine, called the AB370. Sequencing machines have improved wildly since Sanger developed his method. It’s the difference between getting a 4 year-old to read Moby Dick and giving a paragraph each to a bunch of Drama majors and asking them to read at once. The speed is thanks to parallel analysis and high throughput technology. The biggest advances in genome sequencing have been increasing speed and accuracy, resulting in reduction in manpower and cost. This pushed scientists to develop new and better forms of genome sequencing. Unfortunately, it is slow, expensive, and (previously) relies on radioactive materials.
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While this sounds like the lamest game in the world, it works very well for sequencing! Using the information from their interruptions, they can repeat the number they gave you. They have to piece together the information you gave them, for example the 25 th number was 5, the 40 th number was 0, and so on. You leave after a few hours and the group has to figure out the 200-digit number. Each time you are interrupted, you have to start again. Every so often a person interrupts you, and you tell them the single digit you were just thinking and where it is in the sequence of 200. You are reading the digits of the number in your head without making a sound. The tricky part is that the number is 200-digits in length. You are thinking of a number and the group has to guess it. For example, the band is the right under the “A” symbolizes the sequence: “ATGCTC A” Reaction with ddTTP: A T, ATGC T, ATGCTCAGĪll the reactions once run a gel would look something like this (Image by Olwen Reina):Įach band denotes the different lengths code. Reaction with ddCTP: ATG C, ATGCT C, ATGCTCAG Reaction with ddATP: A, ATGCTC A, ATGCTCAG Here is an example where the ddNTPs are in bold and the dNTPs are not: By running the samples on a gel with 4 lanes, you can piece together the sequence as each sequence has been replicated from the same original material. At the end of the PCR, each of your four reactions will yield PCR products of various lengths because replication is randomly terminated. Each reaction contains a with dNTP mix with one of the four nucleotides substituted with a ddNTP (A, T, G, and C ddNTP groups). To perform Sanger Sequencing, you add your primers to a solution containing the genetic information to be sequenced, then divide up the solution into four PCR reactions. When these bases bind to the growing DNA sequence, they terminate replication as they cannot bind other bases. The Sanger sequencing method relies on dideoxynucleotides ( ddNTPs),a type of deoxynucleoside triphosphates (dNTPs), that lack a 3′ hydroxyl group and have a hydrogen atom instead. The Human Genome Project began with Sanger sequencing technology. Since Sanger sequencing (or the chain termination method) is the first generation of sequencing technology, understanding it is greatly important. This is now seen as the “first generation technology” of genome sequencing. His technique used the chain termination method. In that same year, Sanger developed the future backbone of the genome era: DNA sequencing technology. NGS builds upon “first generation sequencing” technologies to yield accurate and cost-effective sequencing results.įred Sanger sequenced the first whole DNA genome, the virus phage ?X174, in 1977. The completion of the Human Genome Project in 2003 ushered in a new era of rapid, affordable, and accurate genome analysis-called Next Generation Sequencing (NGS).