차세대 시퀀싱: DNA 분석 및 진단의 혁신

### 차세대 염기서열 분석(NGS) 기술 및 애플리케이션
* "차세대 염기서열 분석(NGS)은 **수천에서 수백만 개의 DNA 분자를 동시에 염기서열 분석**할 수 있는 강력한 플랫폼입니다."
* NGS는 여러 개인을 동시에 시퀀싱할 수 있는 높은 처리량 옵션을 제공함으로써 **개인 맞춤형 의학, 유전 질환, 임상 진단**과 같은 분야에 혁신을 일으키고 있습니다.
* 1900년대에 처음 개발된 Sanger 시퀀싱은 **DNA 시퀀싱의 황금 표준**으로, 오늘날에도 일상적인 시퀀싱 애플리케이션과 NGS 데이터 검증에 광범위하게 사용되고 있습니다.
* 🔬 Sanger 시퀀싱은 **DNA 사슬 신장을 종결하는 디-디옥시뉴클레오타이드**를 사용하며, 자동 DNA 염기서열 감지를 위해 **고유한 형광 염료**가 있습니다.
* 📊 "지난 10년 동안 도입된 차세대 시퀀싱 시스템은 **수백만 또는 수십억 개의 반응을 동시에 분석**할 수 있는 대규모 병렬 시퀀싱 반응을 가능하게 합니다."
* 모든 NGS 플랫폼은 샘플 준비, 시퀀싱 기계가 필요하며 원시 데이터 출력을 제공하지만, 주로 **시퀀싱 반응의 기술적 세부 사항**에서 차이가 있습니다.
* 💡 NGS 기술에는 **파이로 시퀀싱, 합성에 의한 시퀀싱(일루미나 플랫폼에서 사용), 연결에 의한 시퀀싱 및 이온 반도체 시퀀싱**이 있습니다.
* 🌐 "abm은 **전체 게놈 시퀀싱, 엑솜 시퀀싱, RNA 시퀀싱, 질병 패널 및 레인 대여**를 포함한 광범위한 차세대 시퀀싱 서비스를 제공합니다."

차세대 시퀀싱(NGS)은 높은 처리량의 DNA 시퀀싱을 가능하게 하여 다양한 분야에서 개인 맞춤형 의료 및 진단을 혁신합니다.

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 pyrosequencing 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. Please leave your questions and comments below
and we will answer them as soon as possible. For more information, please visit our knowledge
base at the link provided below. Thank you for watching!

1) Next Generation Sequencing (NGS) - An Introduction

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차세대 염기서열 분석(NGS)의 힘

차세대 시퀀싱(NGS)은 수천에서 수백만 개의 DNA 분자를 동시에 시퀀싱할 수 있게 함으로써 DNA 시퀀싱에 혁명을 일으켰습니다.

높은 처리량으로 개인 맞춤형 의학, 유전 질환, 임상 진단 분야를 혁신하고 있습니다.

오늘날에도 널리 사용되고 있는 생어 시퀀싱은 DNA 의존성 중합효소와 디데옥시뉴클레오타이드를 사용하여 DNA 사슬 신장을 종결하고 자동 DNA 시퀀스 검출을 가능하게 하는 DNA 시퀀싱의 표준으로 사용되고 있습니다.

이 기술은 일상적인 시퀀싱 애플리케이션과 NGS 데이터의 검증에 여전히 중요합니다.

Sanger 시퀀싱은 모세관 기반 전기영동을 사용하여 여러 반응을 분석하는 반면, 차세대 시퀀싱은 대량 병렬 시퀀싱을 가능하게 합니다.

모든 차세대 시퀀싱 플랫폼은 샘플 준비가 필요하고, 시퀀싱 기계를 사용하여 DNA 단편을 증폭하며, 원시 데이터 출력을 제공합니다.

이러한 플랫폼은 기술적 세부 사항이 다르며 파이로 시퀀싱, 합성, 연결, 이온 반도체 시퀀싱으로 분류되며, 각 플랫폼은 시퀀싱 반응을 모니터링하는 고유한 방법이 있습니다.

합성 및 연결에 의한 염기서열 분석 과정

합성에 의한 시퀀싱은 한 번에 하나의 뉴클레오타이드 통합하고 반응이 진행됨에 따라 오류율이 증가하는 Illumina NGS 플랫폼에서 사용됩니다.

반면, 결합에 의한 시퀀싱은 형광 염료가 있는 8-머 올리고뉴클레오타이드 프로브를 사용하여 DNA 중합효소 없이 서로 결합합니다.

이 방법은 프라이머 결합, 프로브 혼성화, 특정 염기에 의해 유도되는 결합에 의존합니다.

그런 다음 이 과정에서 형광 신호가 감지되고 기록됩니다.

파이로 시퀀싱, 이온 반도체 시퀀싱, 연결에 의한 시퀀싱을 포함한 DNA 시퀀싱 기술에는 각각 고유한 프로세스와 장단점이 있습니다.

파이로 시퀀싱은 짧은 시퀀싱 판독을 생성하는 반면, 이온 반도체 시퀀싱은 수소 이온 방출을 감지하며, 연결에 의한 시퀀싱은 Illumina HiSeq에 비해 출력이 제한적입니다.

합성 방법에 의한 시퀀싱은 가장 효율적이며, 높은 커버리지와 여러 개체를 빠르게 시퀀싱할 수 있는 기능을 제공하여 전체 게놈 시퀀싱에 필요한 30배 커버리지 요건을 충족합니다.

이온 양성자 기계는 빠른 처리 시간으로 인해 주로 임상 환경에서 사용됩니다.

ABM은 전체 게놈 시퀀싱, RNA 시퀀싱, 질병 패널을 포함한 광범위한 차세대 시퀀싱 서비스를 제공합니다.

서비스에 액세스하고 자세한 정보를 얻으려면 ABM 웹사이트를 방문하여 "NG 시퀀싱 서비스" 페이지로 이동하여 사용 가능한 서비스, 기술 정보, 가격 및 생물정보학 솔루션에 대한 자세한 내용을 확인하세요.