Next Generation Sequencing (NGS) is a powerful platform 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 sequence multiple individuals at the same time. Sanger Sequencing, first developed in the 1900s, is the gold standard for DNA sequencing and it is still used today extensively for routine sequencing applications and to validate NGS data.
It utilizes a high fidelity DNA-dependent polymerase to generate a complimentary copy to a single stranded DNA template. In each reaction a single primer, complementary to the template, initiates a DNA synthesis reaction from its 3’ end. Deoxynucleotides or simply nucleotides are added one after the other in 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 allow incorporation into the growing strand, however, they differ from natural deoxynucleotides in two ways: 1) they lack a 3’ hydroxyl group which is required for further DNA extension resulting in chain termination once incorporated in the DNA molecule, and 2) each di-deoxynucleotide has a unique fluorescent dye attached to it allowing for automatic detection of the DNA sequence. 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 sequencing machine, either manually onto slab gels or automatically with capillaries, and are electrophoresed to separate the DNA molecules by size. The DNA sequence is read through the fluorescent emission of the di-deoxynucleotide as it flows through the gel. Modern day Sanger Sequencing instruments use capillary based automated electrophoresis, which typically analyzes 8–96 sequencing reactions simultaneously.
Next Generation Sequencing systems have been introduced in the past decade that allow for massively parallel sequencing reactions. These systems are capable of analyzing millions or even billions of sequencing reactions at the same time. Although different machines have been developed with various differing technical details, they all share some common features Sample Preparation: All Next Generation Sequencing platforms require a library obtained either by amplification or ligation with custom adapter sequences.
Sequencing machines: Each library fragment is amplified on a solid surface with covalently attached DNA linkers that hybridize the library adapters. This amplification creates clusters of DNA, each originating from a single library fragment; each cluster will act as an individual sequencing reaction. and, Data output: Each machine provides the raw data at the end of the sequencing run.
This raw data is a collection of DNA sequences that were generated at each cluster. The differences between the different Next Generation Sequencing platforms lie mainly in the technical details of the sequencing reaction and can be categorized in 4 groups: pyrosequencing, sequencing by synthesis, sequencing by ligation, and ion semiconductor sequencing. In pyrosequencing, the sequencing reaction is monitored through the release of a pyrophosphate during each nucleotide incorporation.
The released pyrophosphate is used in a series of chemical reactions resulting in the generation of light. Light emission is detected by a camera which records the appropriate sequence of the cluster. The sequencing proceeds by incubating one base at a time, measuring the light emission (if any), degrading the unincorporated bases, and then the addition of another base.
This technology is capable of generating large read lengths, much comparable to the read length of Sanger Sequencing. However, high reagent cost, and high error rate over strings of 6 or more homopolymers have reduced its applications. For more details on the technical aspect of this technology, please visit our knowledge base at the link provided in the description below.
Sequencing by synthesis utilizes the step-by-step incorporation of reversibly fluorescent and terminated nucleotides for DNA sequencing and is used by the Illumina NGS platforms. All four nucleotides are added to the sequencing chip at the same time and after nucleotide incorporation, the remaining DNA bases are washed away. The fluorescent signal is read at each cluster and recorded; both the fluorescent molecule and the terminator group are then cleaved and washed away.
This process is repeated until the sequencing reaction is complete. This system is able to overcome the disadvantages of the pyrosequencing system by only incorporating a single nucleotide at a time, however, as the sequencing reaction proceeds, the error rate of the machine also increases. This is due to incomplete removal of the fluorescent signal which leads to higher background noise levels.
Our NGS - An Introduction knowledge base provides more technical details about this technology. Sequencing by ligation is different from the other 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 dyes attached to its 5’ end that are ligated to one another.
Each 8-mer consists of two probe specific bases, and six degenerate bases. The sequencing reaction commences by binding of 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 are washed away, the signal is detected and recorded. After that, the fluorescent signal, along with 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 sequencing primer, offset by one base from the previous primer, is used to repeat these steps - in total 5 sequencing primers are used.
The major disadvantage of this technology is the very short sequencing reads generated. Ion semiconductor sequencing utilizes the release 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 changes in the pH of the solution.
During nucleotide incorporation, a single hydrogen ion is released into the solution and it is detected by the semiconductor. The sequencing reaction itself proceeds similarly to pyrosequencing, but at a fraction of the cost. Please view our knowledge base for further details on ion semiconductor sequencing and the sequencing by ligation techniques.
In order to be able to showcase and compare the different technical aspects of each of the above technologies, the number of coverage that 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 most powerful 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. As it can be seen, the pyrosequncing method is only able to sequence the E. coli genome at enough coverage to result in valid data.
The sequencing by synthesis method, which is the most popular method currently on the market, is able to generate hundreds of coverage per run. 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 HiSeq machines. The Ion proton machine is used mostly in clinical setting, because it is able to provide a sufficient size output within 2 hours. abm offers a wide range of Next Generation Sequencing services.
These include whole genome sequencing, exome sequencing, RNA sequencing, disease panels, lane rentals, and much more. To be able to access our services, please visit our website at www. abmgood.
com and from there click on the “NG Sequencing Services” 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 bioinformatics solutions that are related to that particular service.
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