next generation sequencing (ngs) is a powerfulplatform that has enabled the sequencing of thousands to millions of dna molecules simultaneously.this powerful tool is revolutionizing fields such as personalized medicine, genetic diseases,and clinical diagnostics by offering a high throughput option with the capability to sequencemultiple individuals at the same time. sanger sequencing, first developed in the1900s, is the gold standard for dna sequencing and it is still used today extensively forroutine sequencing applications and to validate ngs data. it utilizes a high fidelity dna-dependentpolymerase to generate a complimentary copy to a single stranded dna template. in eachreaction a single primer, complementary to the template, initiates a dna synthesis reactionfrom its 3’ end. deoxynucleotides or simply
nucleotides are added one after the otherin a template-dependent manner. each reaction also contains a mixture of four di-deoxynucleotides,one for each dna base. these di-deoxynucleotides resemble the dna monomers enough to allowincorporation into the growing strand, however, they differ from natural deoxynucleotidesin two ways: 1) they lack a 3’ hydroxyl group which is required for further dna extensionresulting in chain termination once incorporated in the dna molecule, and 2) each di-deoxynucleotidehas a unique fluorescent dye attached to it allowing for automatic detection of the dnasequence. as a result many copies of different-length dna fragments are generated in each reaction,terminated at all of the nucleotide positions of the template molecule by one of the di-deoxynucleotides.
the reaction mixtures are loaded on the sequencingmachine, either manually onto slab gels or automatically with capillaries, and are electrophoresedto separate the dna molecules by size. the dna sequence is read through the fluorescentemission of the di-deoxynucleotide as it flows through the gel. modern day sanger sequencinginstruments use capillary based automated electrophoresis, which typically analyzes8–96 sequencing reactions simultaneously. next generation sequencing systems have beenintroduced in the past decade that allow for massively parallel sequencing reactions. thesesystems are capable of analyzing millions or even billions of sequencing reactions atthe same time. although different machines have been developed with various differingtechnical details, they all share some common
features sample preparation: all next generation sequencingplatforms require a library obtained either by amplification or ligation with custom adaptersequences. sequencing machines: each library fragmentis amplified on a solid surface with covalently attached dna linkers that hybridize the libraryadapters. this amplification creates clusters of dna, each originating from a single libraryfragment; each cluster will act as an individual sequencing reaction. and, data output: each machine provides theraw data at the end of the sequencing run. this raw data is a collection of dna sequencesthat were generated at each cluster.
the differences between the different nextgeneration sequencing platforms lie mainly in the technical details of the sequencingreaction and can be categorized in 4 groups: pyrosequencing, sequencing by synthesis, sequencingby ligation, and ion semiconductor sequencing. in pyrosequencing, the sequencing reactionis monitored through the release of a pyrophosphate during each nucleotide incorporation. thereleased pyrophosphate is used in a series of chemical reactions resulting in the generationof light. light emission is detected by a camera which records the appropriate sequenceof the cluster. the sequencing proceeds by incubating one base at a time, measuring thelight emission (if any), degrading the unincorporated bases, and then the addition of another base.
this technology is capable of generating largeread lengths, much comparable to the read length of sanger sequencing. however, highreagent cost, and high error rate over strings of 6 or more homopolymers have reduced itsapplications. for more details on the technical aspect of this technology, please visit ourknowledge base at the link provided in the description below. sequencing by synthesis utilizes the step-by-stepincorporation of reversibly fluorescent and terminated nucleotides for dna sequencingand is used by the illumina ngs platforms. all four nucleotides are added to the sequencingchip at the same time and after nucleotide incorporation, the remaining dna bases arewashed away. the fluorescent signal is read
at each cluster and recorded; both the fluorescentmolecule and the terminator group are then cleaved and washed away. this process is repeateduntil the sequencing reaction is complete. this system is able to overcome the disadvantagesof the pyrosequencing system by only incorporating a single nucleotide at a time, however, asthe sequencing reaction proceeds, the error rate of the machine also increases. this isdue to incomplete removal of the fluorescent signal which leads to higher background noiselevels. our ngs - an introduction knowledge base provides more technical details aboutthis technology. sequencing by ligation is different from theother two methods since it does not utilize a dna polymerase to incorporate nucleotides.instead, it relies on 16 8-mer oligonucleotide
probes, each with one of 4 fluorescent dyesattached to its 5’ end that are ligated to one another. each 8-mer consists of twoprobe specific bases, and six degenerate bases. the sequencing reaction commences by bindingof the primer to the adapter sequence and then hybridization of the appropriate probe.this hybridization of the probe is guided by the two probe specific bases and upon annealing,is ligated to the primer sequence through a dna ligase. unbound oligonucleotides arewashed away, the signal is detected and recorded. after that, the fluorescent signal, alongwith the last 3 bases of the 8-mer probe, are cleaved, and then the next cycle commences.after approximately 7 cycles of ligation the dna strand is denatured and another sequencingprimer, offset by one base from the previous
primer, is used to repeat these steps - intotal 5 sequencing primers are used. the major disadvantage of this technologyis the very short sequencing reads generated. ion semiconductor sequencing utilizes therelease of hydrogen ions during the sequencing reaction to detect the sequence of a cluster.each cluster is located directly above a semiconductor transistor which is capable of detecting changesin the ph of the solution. during nucleotide incorporation, a single hydrogen ion is releasedinto the solution and it is detected by the semiconductor. the sequencing reaction itselfproceeds similarly to pyrosequencing, but at a fraction of the cost. please view ourknowledge base for further details on ion semiconductor sequencing and the sequencingby ligation techniques.
in order to be able to showcase and comparethe different technical aspects of each of the above technologies, the number of coveragethat each run generates when sequencing the whole human, mouse, arabidopsis thaliana,and e. coli genome are calculated and presented here. the presented data is based on the mostpowerful machines of each technology, further details can be found on our knowledge base.for whole genome sequencing data to be useful a minimum of 30x coverage is required. asit can be seen, the pyrosequncing method is only able to sequence the e. coli genome atenough coverage to result in valid data. the sequencing by synthesis method, which is themost popular method currently on the market, is able to generate hundreds of coverage perrun. in fact, with this machine it is possible
to sequence 15 individuals within 3.5 days.the sequencing by ligation method also generates enough coverage for all genomes to be used,however, it isn’t capable of generating nearly as much output as the illumina hiseqmachines. the ion proton machine is used mostly in clinical setting, because it is able toprovide a sufficient size output within 2 hours. abm offers a wide range of next generationsequencing services. these include whole genome sequencing, exome sequencing, rna sequencing,disease panels, lane rentals, and much more. to be able to access our services, pleasevisit our website at www.abmgood.com and from there click on the “ng sequencing servicesâ€link. this will load our ngs service webpage
which details all of our available services.clicking on a service of interest will showcase the technical details, pricing, and bioinformaticssolutions that are related to that particular service. please leave your questions and comments belowand we will answer them as soon as possible. for more information please visit our knowledgebase at the link provided below. thank you for watching!
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