1

NEXT
GENERATION
SEQUENCING
(NGS)
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TABLE OF CONTENTS

DEFINITION ............................................................................................................................. 4

HISTORY .................................................................................................................................. 4

PRINCIPLE ............................................................................................................................... 5

1. Fragmentation ................................................................................................................. 5

2. Adaptation ....................................................................................................................... 5

3. Amplification .................................................................................................................. 6

4. Data Analysis .................................................................................................................. 6

DIFFERENCES BETWEEN NGS AND SANGER SEQUENCING ....................................... 6

PROCEDURE ............................................................................................................................ 7

Second Generation Sequencing ............................................................................................. 7

• Illumina Sequencing:................................................................................................... 7

• 454 Sequencing (Roche Diagnostics): ........................................................................ 8

• Ion Torrent Sequencing: .............................................................................................. 9

Third Generation Sequencing ................................................................................................ 9

• PacBio Sequencing (Pacific Biosciences): .................................................................. 9

Fourth Generation Sequencing ............................................................................................ 10

• SOLiD Sequencing:................................................................................................... 10

PROGRESS THANKS TO NGS ............................................................................................. 10

1) Accessible Whole-Genome Sequencing ................................................................... 10

2) Broad Dynamic Range for Expression Profiling....................................................... 10

3) Tunable Resolution for Targeted NGS...................................................................... 11

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COMPARISON OF SANGER SEQUENCING AND NGS ................................................... 11

Benefits ................................................................................................................................ 11

Challenges ............................................................................................................................ 11

SCHEME OF NEXT GENERATION SEQUENCING .......................................................... 12

APPLICATIONS ..................................................................................................................... 13

1. Genomic Techniques .................................................................................................... 13

2. Genomic Analysis ......................................................................................................... 13

3. Variant Detection .......................................................................................................... 14

4. Identifying De Novo Mutations .................................................................................... 14

5. Somatic Variant Detection ............................................................................................ 14

6. Testing Hereditary Disorders ........................................................................................ 15

7. Identification of Secondary Mutations.......................................................................... 15

8. Detecting Microbial Organisms .................................................................................... 15

9. Applications in Oncology ............................................................................................. 16

10. Familial Genetic Testing ........................................................................................... 16

11. Metagenomics ........................................................................................................... 17

NGS IN PRACTICE ................................................................................................................ 17

• Whole-Exome Sequencing............................................................................................ 17

• Targeted Sequencing ..................................................................................................... 18

IMPACT OF NGS ON GENETICS ........................................................................................ 18

CHALLENGES: ...................................................................................................................... 18

LIMITATIONS ........................................................................................................................ 19

THE FUTURE ......................................................................................................................... 19

CONCLUSION ........................................................................................................................ 20

REFERENCES ........................................................................................................................ 21
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NEXT GENERATION SEQUENCING

(NGS)

DEFINITION

Next Generation Sequencing (NGS) is an advanced technology for DNA Sequencing. It is

much faster than old Sanger Sequencing, i.e., it can sequence whole human genome in one day.

Next-generation sequencing (NGS) is a massively parallel sequencing technology that offers

ultra-high data, scalability, and speed. The technology is used to determine the order of
nucleotides in entire genomes or targeted regions of DNA or RNA. Today's complicated
genomics problems need a degree of information that conventional DNA sequencing
technology cannot provide. NGS has filled that void and has revolutionized the biological
sciences, enabling labs to undertake a wide range of applications and research biological
systems at a seemingly impossible complexity.

HISTORY

In 1977, ground-breaking articles describing methods for DNA sequencing were published.
Allan Maxam and Walter Gilbert reported an approach in which terminally labeled DNA
fragments were subjected to base specific chemical cleavage and the reaction products were
separated by gel electrophoresis. In an alternative approach, Frederick Sanger and colleagues
described the use of chain-terminating dideoxy-nucleotide analogs that caused base-specific
termination of primed DNA synthesis. Because of its high efficiency and low radioactivity,
Sanger sequencing was adopted as the core technology in the “First Generation” of laboratory
and commercial sequencing applications. At that time, DNA sequencing was tough and
radioactive materials were required.

Applied Biosystems introduced the first automatic sequencing machine in 1987, employing
capillary electrophoresis, which made the sequencing faster and more accurate. This could
detect 96 bases in a one cycle, 500 K bases a day, and read lengths of up to 600 bases. Since
1995, the latest model has been capable of producing 2.88 million bases per day and read
lengths of up to 900 bases.

In an industrial, high-throughput configuration, Sanger technology was used in the sequencing

of the first human genome, which was completed in 2003 through the Human Genome Project,
a 13-year effort with an estimated cost of $2.7 billion. This project greatly stimulated the
development of powerful novel sequencing instrument to increase speed and accuracy, while
simultaneously reducing cost and manpower.
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In 2008, by comparison, a human genome was sequenced over a 5-month period for
approximately $1.5 million. The latter accomplishment highlights the capabilities of the rapidly
evolving field of “Next Generation Sequencing” (NGS) technologies that have emerged during
the past few years.

PRINCIPLE

DNA fragments are sequenced and analyzed. To sequence the complete human genome, the
data of several segments must be put together, which is both time-consuming and expensive.
The method is controlled using Next Generation Sequencing, and the results are acquired in
sync with the sequencing. The results can also be compared to a human reference genome. As
a result, the NGS is both quicker and less expensive.

[FIGURE: Basic principles of sequencing chemistry. (a) Normal chain elongation leads to the release of (b)
pyrophosphate (Roche 454) and (c) a hydrogen ion (Ion Torrent). (d) Structure of the ddNTP that forms the basis
of Sanger sequencing. (e) Sketch of the phosphor-linked fluorophore (PacBio). (f) Sequencing by ligation
(SOLiD). (g) Sketch of reversible chain terminators (Illumina and Helicos)]

The basic principle of the NGS is based on four steps:

1. Fragmentation

In the first step, with the help of enzymes or by centrifugation, DNA is cut into
fragments are generated.

2. Adaptation

In the next step, certain adapter oligonucleotides are bound to the fragment to create a
"DNA library".
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3. Amplification

DNA fragments are bound to a solid reaction medium and replicated. The same DNA
cluster will be generated where the actual sequence will take place. Because it is divided
into clusters, many sequence operations can be performed simultaneously in a very
short time.

4. Data Analysis

The generated data is saved in the form of a DNA chip and bioinformatically evaluated.
Data volumes of up to 200 GB are possible. This makes storing data and, more
importantly, passing it on to other scientists or clinicians more problematic. As a result,
many laboratories use cloud services.

DIFFERENCES BETWEEN NGS AND SANGER SEQUENCING

Major advancements include the use of fluorescent-labeled nucleotides instead of radioactivity,

the replacement of capillary electrophoresis for gel electrophoresis, and the enhancement of
DNA polymerases.

The concepts behind Sanger vs. Next-Generation Sequencing (NGS) technologies are similar
in general. In both NGS and Sanger sequencing, DNA polymerase sequentially adds
fluorescent nucleotides to a growing DNA template strand. The fluorescent tag on each
integrated nucleotide identifies it.

[FIGURE Sanger Sequencing Mechanism]

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The key distinction between Sanger sequencing and NGS is the amount of data sequenced.
While the Sanger method only sequences a single DNA fragment at a time, NGS is massively
parallel and may sequence millions of fragments at the same time per run. This high-throughput
method allows for the simultaneous sequencing of hundreds to thousands of genes.

PROCEDURE

Previous gene sequencing approaches relied on enzymatic (Sanger sequencing) or chemical

(Maxam-Gilbert Method) procedures. NGS utilizes several techniques. Scope is essential in
selecting on a method. It indicates the bare minimum of reads needed to create a reference
sequence. A read is a base sequence that matches the base sequence of a DNA fragment.

The phrase "next generation" has indicated a next step in the evolution of DNA sequencing
technology and implies that new technologies will be named "next-next" generation in the
future, so the terms second generation, third generation, and so on are preferred.

Second Generation Sequencing

• Illumina Sequencing:
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[FIGURE: The procedure of the Illumina Genome Analyzer is depicted here. Before applying the library
to the solid surface of a flow cell, similar fragmentation and adapter ligation processes are performed (I).
Attached DNA fragments create 'bridge' molecules, which are then amplified via isothermal
amplification, resulting in a cluster of identical fragments that are then denatured for sequencing primer
annealing (II). Sequencing-by-synthesis is performed on amplified DNA fragments utilizing 30 blocked
labelled nucleotides (III).]

Adapter-ligated, single-stranded sequences are annealed to a glass plate precoated with

oligonucleotides corresponding to the adapters after DNA fragmentation, adapter
ligation, and gel purification. These oligos act as both template DNA capture and
primers for further amplification. Amplification happens on the slide by a mechanism
known as 'bridge amplification,' in which each single-stranded molecule binds to the
oligo primers on the slide at both ends.
Following rounds of PCR, little islands or clusters of amplified molecules are formed,
which serve as clones for future sequencing using chain terminators, like standard
Sanger sequencing. Unlike the Sanger approach, however, Illumina employs
fluorescently labelled reversible terminators, such that each single base inclusion on
each molecule momentarily stops the synthesis. To identify which nucleotide is
integrated in each DNA clonal cluster, a high-resolution digital picture is employed.
Following imaging, the terminator is chemically reversed, allowing the template
molecule to be expanded again in the following cycle of sequencing. As a result, the
sequencing reaction completes on the vast majority of molecules.

• 454 Sequencing (Roche Diagnostics):

Roche 454 was the first commercially successful next generation system. This
sequencer uses pyrosequencing technology instead of dideoxy nucleotides.
Pyrosequencing is based on the detection of pyrophosphate, which is produced during
nucleotide incorporation.
The library DNAs containing 454-specific adaptors are denatured into single strands
and captured using amplification beads before being subjected to emulsion PCR. If a
base pairing occurs, pyrophosphate is produced, which is then transformed to adenosine
triphosphate via an enzyme process. Then, using ATP sulfurylase, luciferase, luciferin,
DNA polymerase, and adenosine 5 phosphosulfate (APS), one of the dNTPs (dATP,
dGTP, dCTP, dTTP) would complement to the bases of the template strand and release
pyrophosphate (PPi) equivalent to the amount of incorporated nucleotide. The ATP
generated by the PPi transformation drives the luciferin into oxyluciferin and creates
visible light. At the same time, apyrase degrades the mismatched bases. The reaction
system is then replenished with another dNTP, and the pyrosequencing procedure is
repeated.
The Roche method has a read length of about 500 bases, making it ideal for amplicon
sequencing and bridging over complicated sequences. Its Achilles' heel is its reliance
on pyrosequencing, which makes sequencing homopolymer runs problematic,
necessitating the use of Roche's 454 sequencing equipment.
The most notable benefit of Roche 454 is its speed: it takes only 10 hours from start to
finish. When compared to other NGS systems, the read length is also notable. However,
the high cost of reagents, which averages $12.56 per 6-10base, continues to be a
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challenge for Roche. Its library creation can be automated, and emulsion PCR may be
computer aided, reducing labor significantly.

• Ion Torrent Sequencing:

A hydrogen ion is released as part of the normal chemical reactions of nucleotide

incorporation in Ion Torrent sequencing. An ion-sensitive semiconductor detects this
ion as a minor change in pH.
The magnitude of the change in pH using Ion Torrent sequencing is determined by the
number of base incorporations, similar to pyrosequencing, which measures released
PPi, making it susceptible to misreading the length of homopolymers.

Third Generation Sequencing

Very long fragments of DNA, up to 30–50 kb, or longer are sequenced with the help of PacBio
sequencing, also known as SMRT (Singe Molecule Real Time) sequencing. The SMRT
approach requires attaching a designed DNA polymerase to the bottom of a well with bound
DNA to be sequenced.

• PacBio Sequencing (Pacific Biosciences):

Unlike previous NGS methods, the DNA polymerase is immobilized on the floor of a
microcell in this approach. To produce circular DNA, fragmented dsDNA is ligated to
hairpin adapters. These are amplified linearly using primers corresponding to the
hairpin sequence, then caught by a single molecule of DNA polymerase and sequenced
at the bottom of a well. Fluorescently labelled nucleotides (one color for each base)
diffuse into the cell from above. Unlike in previous systems, the fluorescent molecule
is phosphor-linked, which means it is cleaved upon incorporation. As these tagged
nucleotides disperse around the polymerase active site, they emit a modest noise signal.
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When the DNA polymerase comes across a nucleotide that is complementary to the
next base in the template, it incorporates it into the developing DNA chain and keeps it
there for orders of magnitude longer than the typical drifting nucleotide. This produces
a definable colored signal that can be distinguished from ordinary diffusion. The
fluorescent label is cleaved and diffuses away after incorporation, allowing the DNA
polymerase to continue incorporating multiple bases per second.

Fourth Generation Sequencing

Long DNA molecules can be passed through tiny diameter "holes" and varying currents
measured as each nucleotide passes by a coupled detector. More than a hundred kb of DNA
could theoretically be passed through the nanopore, and with numerous channels, tens to
hundreds of GB of sequence may be obtained at a minimal cost. One of the examples include
OXFORD Nanopore Technology (minION).

• SOLiD Sequencing:

Sequencing, unlike any other method, is accomplished by hybridization and ligation.

DNA fragmentation is followed by ligation to beads and emulsion PCR. The 39
dinucleotides, of which there are precisely 16 combinations, provide ligation and
sequencing specificity. 16 probes are labelled with one of four dyes, leaving any
particular dye connected with four primers.

PROGRESS THANKS TO NGS

1) Accessible Whole-Genome Sequencing

The Human Genome Project took over ten years and cost approximately $3 billion to
complete using capillary electrophoresis-based Sanger sequencing. In contrast, next-
generation sequencing (NGS) makes large-scale whole-genome sequencing (WGS)
affordable and feasible for the regular researcher. It enables scientists to sequence
thousands to tens of thousands of genomes in a single year, or to study the whole human
genome in a single sequencing run.

2) Broad Dynamic Range for Expression Profiling

NGS-based RNA-Sequencing is a strong technology that allows researchers to

overcome the inefficiency and high cost of outdated technologies such as microarrays.
Disturbance at the low end and signal saturation at the high-end limit microarray gene
expression measurement. Next-generation sequencing, on the other hand, quantifies
distinct, digital sequencing read counts, providing a wider dynamic range.
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3) Tunable Resolution for Targeted NGS

Targeted sequencing allows you to sequence several genes or specific genomic regions
of interest, emphasizing the ability of NGS in an efficient and cost-effective manner.
NGS is extremely scalable, allowing you to tune the level of resolution to match the
needs of your experiments. Choose between a basic scan across several samples or a
detailed sequencing with fewer samples to find unusual variants in a particular location.

COMPARISON OF SANGER SEQUENCING AND NGS

Sanger Sequencing Targeted NGS

• Fast • Higher sequencing depth allows

• Cost-effective sequencing for for greater sensitivity (down to 1
low numbers of targets (1–20 percent)
targets) • Greater ability to discover
Benefits • Improved mutation resolution
• With the same amount of input
DNA, more info is created.
• Increased sampling throughput

• Low sensitivity • Less cost-effective for sequencing

• Low discovery power low numbers of targets (1–20
• Not as cost-effective for high targets)
Challenges numbers of targets (> 20 targets) • Time-consuming for sequencing
• Low scalability due to increasing low numbers of targets (1–20
sample input requirements targets)
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SCHEME OF NEXT GENERATION SEQUENCING

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APPLICATIONS

Innovative sample preparation and data analysis options enable a broad range of applications.
For example, NGS allows labs to:

• Use RNA sequencing (RNA-Seq) to find novel RNA variations and splice sites, or to
quantify mRNAs for gene expression research.
• Examine epigenetic variables such as genome-wide DNA methylation and DNA-
protein interactions
• Sequence cancer samples to investigate unusual somatic variations, tumor subclones,
and other topics.
• Investigate the human microbiota
• Discover new pathogens

Some of the applications are discussed below:

1. Genomic Techniques

There are two techniques to using NGS to investigate specific sections of the genome.
The first method is by PCR, which often involves numerous primer pairs in a mixture
that are coupled with genomic DNA of interest in a multiplex technique to retain
valuable DNA. Following the PCR, platform-specific adapters are ligated to the ends
of the resultant fragments to generate a library appropriate for sequencing. The second
includes hybrid capture, which has been developed and marketed by numerous groups.
This exploits the hybridization of DNA fragments from a whole-genome library to
complementary sequences produced and concatenated. When the probes are designed
to catch almost all of the known coding exons in a genome, the capture method is known
as "exome sequencing."

2. Genomic Analysis

The rate of advancement in analytical approaches to genome-wide data analysis

remains rapid. The digital character of NGS data is critical to many analytical
methodologies. The library's DNA fragments are amplified on either beads or flat
surfaces. Each sequence read corresponds to a single DNA fragment. Methods for
finding novel disease genes have been driven by technological breakthroughs. To
uncover the genetic origins of a trait, early research relied on families in which a disease
was segregating. For highly penetrant, monogenic disorders like cystic fibrosis, these
linkage analysis investigations were effective. Standard parametric linkage analyses of
several complex characteristics were successful, especially when samples were drawn
from the extreme ends of the phenotypic range. For example, studying families with
early-onset Alzheimer's disease resulted in the identification of numerous genes that
contribute significantly to the phenotypic and gave light on the molecular processes
(e.g., plaque formation) of disease development.
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3. Variant Detection

With the dawn of NGS, it is now possible to investigate almost every base in the
genome, and approaches for accurately interpreting and identifying millions of
variations are being developed. The advantage of sequencing in this regard is that most
variants, both common and rare, can be discovered with sufficient sequencing read
coverage, algorithmic methods to identify the variants, and careful analogous validation
to distinguish true from false positives. The exception to this research ability is the
reliance on conformity to the Human Genomic Reference sequence, which is the first
stage in NGS data analysis, because this reference does not contain the entire unique
genome content across all individuals. For the identification and sequencing of
germline SNPs and minor insertions and deletions in high-throughput sequencing data,
many variant calling techniques have been developed. Once identified, these variations
may be studied in case-control studies utilizing the same methodologies established for
Genome Wide Association Studies (GWAS).

4. Identifying De Novo Mutations

De novo mutations, or variants that appear for the first time in an individual, are the
most unusual. They are extremely important in systems biology because they are more
likely to have functional effects in rare diseases. Characterizing these mutations also
provides for an estimate of the baseline human mutation rate and its relationship to
parental age. Because sequencing reads have a larger error margin than traditional
sequencing, these tools include coverage, sequencing error rate, predicted de novo
mutation rate, and family ties.

5. Somatic Variant Detection

The comparison of a person's cancer genome to the normal genome (taken from
unaffected tissue DNA) offers a thorough description of the somatic alterations that
occur throughout the transformation from normal to malignant cells. In comparison to
exome sequencing, whole genome sequencing (WGS) techniques to somatic variant
identification are more difficult owing to the bulk of the data and the multiple types of
variations that may be detected using different algorithmic predictors. However,
morphological changes, being the most challenging to predict accurately and with a
good accuracy, are common in cancer genomes and can only be detected using WGS
data. With a growing emphasis on identifying cancer heterogeneity, the capacity of
somatic variant detection algorithms to anticipate low-frequency single-nucleotide
variations are becoming increasingly important.
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6. Testing Hereditary Disorders

Next-generation sequencing has been useful in determining the underlying molecular

cause of diseases, particularly those that are genetically diverse. Usher syndrome is an
autosomal recessive condition with deafness, retinitis pigmentosa, and vestibular
disorders. NGS has enabled the creation of tailored gene panels to investigate all causal
genes related with Usher Syndrome. Previously, a thorough investigation was hindered
by the labor-intensive and expensive expense of Sanger sequencing. Providing an
earlier diagnosis for individuals with Usher Syndrome allows patients and their families
to begin medical treatment sooner.

7. Identification of Secondary Mutations

The discovery of secondary mutations as causes of resistance to certain medicines is

another key application of massively parallel sequencing due to its capacity to deeply
scan specific genomic areas. Several lines of evidence show that secondary mutations
in target genes cause de novo and acquired resistance to some targeted therapies (for
example, the T790M mutation in the EGFR gene causes resistance to anti-epidermal
growth factor receptor agents) or in genes whose inactivation is synthetically fatal in
the presence of the therapeutic applications.

8. Detecting Microbial Organisms

NGS has been shown to be useful in a variety of areas, including pathogen biology and
genomic epidemiology. Targeted sequencing and unbiased interrogation of clinical
samples for pathogen detection and identification, drug resistance profiling, strain
typing and epidemiological outbreak investigation, microbiome studies, genomic
determinant analysis of microbial functions including metabolism, and comparative
ribosomal RNA phylogenetic studies are examples of these. When previous techniques
fail to identify an organism or cannot decode complex specimens, such as in patients
with polymicrobial infections, next-generation sequencing of pathogen biology and
genomic epidemiology provides a viable diagnostic alternative. For pathogens that have
not yet been thoroughly defined, NGS can undertake complete de novo genome
sequencing, giving reference genomes for subsequent research. The use of NGS to
detect Leptospira in a critically ill young patient's cerebral spinal fluid was a pivotal
event that established the clinical value of honest and unbiased NGS. To make NGS an
effective or complementary tool for routine use in clinical microbiology, improvements
are required.
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9. Applications in Oncology

Next-generation sequencing tests have been used to diagnose and manage oncology
patients since the technique was first used to diagnose individuals with solid tumors or
hematologic abnormalities. Next-generation sequencing, also known as Whole Genome
Sequencing (WGS) and Whole Exome Sequencing (WES), is capable of detecting a
wide range of genetic/somatic variations in cancer. NGS can detect either germline or
somatic variations, depending on the purpose of the test. The advantage of NGS is its
capacity to conduct large-scale, inclusive, and sensitive searches for numerous
sequence variants. When compared to several nucleic acid-based tests (such as
fluorescence in situ hybridization, PCR/sequencing, and so on), the technology can
actually save expenditure. Next-generation sequencing has been utilized as a molecular
diagnostic tool for a variety of solid tumor and hematologic cancers. Test results can
assist in the initial diagnosis, tumor categorization, pinpointing the origin of the
malignancy, and determining the outcome. Future advancements will make NGS
technology more inexpensive, allowing for broader applications such as circulating
tumor DNA detection or liquid biopsy. Thyroid nodules are an example of a situation
in which fine needle aspiration and cytologic examination may not provide a conclusive
diagnosis, whereas NGS has been demonstrated to have higher sensitivity and
specificity for cancer detection. However, not all patients will gain clinically enough to
afford the price of NGS testing, therefore testing should be used with caution. The
initial cost of purchasing the equipment, as well as complicated bioinformatics to
understand the sequencing data, are among the present barriers limiting wider
implementation.

10. Familial Genetic Testing

Individuals assessed to be at high risk owing to their family and clinical history are
given genetic testing for high penetrance familial cancer genes. The use of NGS could
result in significant cost savings due to the simultaneous sequencing of numerous
targets and samples. Individuals with diseases such as breast and ovarian cancer who
may not meet the existing severe criteria for recommending genetic testing may become
eligible for screening because of reduced-cost sequencing. A conventional genetic
screen could contain lower frequency genes that are not routinely screened but are
implicated in familial cancer disorders. For example, poly (ADP ribose) polymerase
(PARP) inhibitor trials and surgical management decisions could benefit from a more
easily accessible enzyme. The use of NGS could result in significant cost savings due
to the simultaneous sequencing of numerous targets and samples. Lower frequency
genes that are not regularly checked but have been linked to familial cancer syndromes
should be included in a classical genetic screen if disease risks can be linked to
mutations in such genes. NGS for breast, ovarian, and serous ovarian cancer could
benefit from studies with poly (ADP ribose) polymerase (PARP) inhibitors and as an
assistance to surgical management decisions. Individuals with diseases such as breast
and ovarian cancer who may not meet the present stringent criteria for recommending
genetic testing may become eligible for screening, as cost is a major component in
deciding the stringency of such criteria.
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11. Metagenomics

The use of next-generation sequencing (NGS) has had a significant impact on the
research of microbial diversity in environmental and clinical samples. In practice,
genomic DNA is extracted from an interest sample, transformed to an NGS library, and
sequenced. The resulting sequence is aligned to known reference sequences for
microorganisms estimated to be present in the sample. Closely related species can be
identified, while distantly related species can be deduced. Furthermore, point mutation
original dataset compilation can offer information to support the presence of recognized
and potentially novel species. Qualitative genomic information is collected, and
quantitative information on particular microbial species can be derived by analyzing
the relative abundance of sequence reads. To date, most NGS-based metagenomic
investigations have relied on 454 technology and longer read lengths to permit
alignment to microbial reference genomes and de novo assembly of previously known
microbial genomes. Metagenomic investigations have included the examination of
microbial populations in the ocean and soil, the discovery of a new arenavirus in
transplant recipients, and the characterization of microflora in the human oral cavity
and the stomachs of fat and lean twins.

The term "RNA-Seq" refers to a powerful new method for mapping and measuring
transcripts in biological materials. Isolated total, ribosomal RNA–depleted, or poly(A)
RNA is processed to cDNA. A common procedure is generating first strand cDNA
using random hexamer–primed reverse transcription. After that, the cDNA is
fragmented and ligated to NGS adapters. Following sequencing, reads are either
matched to a reference genome, compared to known transcript sequences, or assembled
from scratch to create a genome-scale transcription map. RNA-Seq has enhanced its
performance in the quantitative detection of both highly generated transcripts and low-
level transcripts. Small RNAs, including as microRNAs (miRNAs) and short
interfering RNAs, are usually isolated using a small RNA– enrichment method, size
selection on an electrophoresis gel, or a combination of these methods. RNA-Seq has
been used to study a wide range of organisms, including Saccharomyces cerevisiae,
Arabidopsis thaliana, mice, and human cells.

NGS IN PRACTICE

• Whole-Exome Sequencing

Clinicians may now discover the genetic origin of an illness in a rapid, cost-effective,
and comprehensive manner thanks to next-generation sequencing (NGS). Exome
sequencing accounts for slightly more than 1% of the genome and is thus significantly
less expensive to sequence than the full genome. NGS can also aid in the identification
of disease-causing mutations in pathogenic manifestations where the specific genetic
origin is unknown. Although high-throughput sequencing of the complete human
genome is possible, researchers and physicians are usually primarily interested in the
protein-coding sections of the genome, known as the exome.
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• Targeted Sequencing

Although whole-genome and whole-exome sequencing are possible, targeting

individual genes or genomic areas is preferred. Targeted sequencing is less expensive,
provides significantly more coverage of genetic areas of interest, and saves sequencing
cost and time. Researchers have started to create sequencing panels that target genomic
areas known to be centers for disease-causing mutations. These panels sequence just
the required areas of the genome, excluding the rest of the genome from study. Targeted
sequencing, whether of individual genes or entire panels of genomic areas, assists in
the early detection of numerous hereditary illnesses.

IMPACT OF NGS ON GENETICS

If one believes that the primary goal of genetics is to discover the genotypes that explain
phenotypes, the parabolic growth in DNA sequence information applied to such an objective
will only go higher. The recent development of technologies capable of produce millions of
DNA sequence reads in a single run is rapidly revolutionizing the world of genetics, giving
previously unthinkable speed in resolving problems. As an endpoint to applications ranging
from chromatin immunoprecipitation, mutation mapping, and polymorphism identification to
noncoding RNA discovery, these technologies will enable an inexpensive genome-wide
sequence readout.

“It became obvious how hit-and-miss gene association studies were in identifying variants.
They were more like fishing expeditions. We realized that NGS would enable us to look at
much larger portions of the genome simultaneously.”

Linda S. Pescatello

(Distinguished Professor of Kinesiology, University of Connecticut)

CHALLENGES:

• It should be mentioned, however, that the massive amount of data generated by next-
generation sequencing research may take a long time to be transformed into clinically
meaningful information.
• Given that each cancer genome may contain more than 10,000 somatic mutations, it is
unclear how much confirmation through recurrent mutation identification or laborious
functional studies will be required to distinguish driver mutations (those that either
confer a tumor's growth/survival advantage or are required for cancer cells to maintain
their malignant behavior) from passenger alterations.
• Furthermore, next-generation sequencing is expected to reveal a far greater complexity
of the normal human genome in terms of SNPs and polymorphisms, some of which
may be restricted to certain somatic organs within the same individual.
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• Massively parallel sequencing will demand the availability of high-performance

computer and bioinformatics assistance far beyond the capabilities of most research
institutions.
• Furthermore, quality control and regulation of massively parallel sequencing
procedures, as well as data reporting, are critical considerations.
• Finally, the ethical concerns of next-generation sequencing are far from trivial.

LIMITATIONS

• NGS costs much less time and money than first-generation sequences, but it's still too
expensive for many labs. To make NGS cost-effective, a high number of sample batches
must be conducted, which may need ultra-regional centralization. Following the first
capital expenditure, the NGS facility's capabilities will be able to deliver national-level
services that are anticipated to generate economic advantages in addition to enhancing
patient care.
• Inaccurate sequences of homopolymer regions (spans of repeating nucleotides) on
certain NGS platforms, including the Ion Torrent PGM, and short sequence read lengths
(average 200-500 nucleotides) can lead to sequence errors.
• The main drawback of NGS in a clinical environment is not only to provide the
necessary setups such as computer capacity and storage space, but also to provide the
personal expertise needed to comprehensively analyze and interpret follow-up data.
• Furthermore, the volume of data must be intelligently managed in order to extract
clinically essential information with a simple and robust interface.

THE FUTURE

Despite the incredible progress achieved in these technologies over the last few decades,
additional innovations are yet possible. There has been a bit of hype in recent years about
single-molecule nanopore technologies, which aspire to provide easy, low-cost single-
molecule DNA sequencing in devices no bigger than a smartphone. These technologies
function by drawing DNA through microscopic pores and reading the bases as they pass as
changes in electric current across the pore. This technique might eliminate the requirement for
enzymatic activities and imaging entirely, and it has the ability to create very lengthy readings
very quickly. While no major data has been collected, this approach may become accessible in
2013. If the technique works as expected, gadgets might be accessible in clinics very soon,
becoming an important tool in diagnostic medicine.
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CONCLUSION

One may argue that highly parallel sequencing is not just a goal in itself, but also a way of
carrying out experiments that may answer questions that could not previously be answered.
The change that Next Generation Sequencing technologies are anticipated to bring about is
similar to the revolution brought about by the advent of the PCR in the 1980s. This technique
will undoubtedly represent a major breakthrough in breast cancer fundamental and translational
research; nonetheless, several hurdles lay ahead. We should learn from our previous experience
with microarrays and avoid unreasonable anticipation. The greatest risk of applying this
revolutionary technology to clinical and translational questions is that it introduces new
problems; if we move too quickly, the lessons we are beginning to learn from historic record
studies may be forgotten when massively parallel sequencing is applied to research and clinical
issues.

“With Sanger sequencing, we saw a limited DNA snapshot… NGS and its massively parallel
sequencing enable us to look at tens to hundreds of thousands of reads per sample.”

Michael Bunce,

(Professor, Head of TrEnD laboratory, Curtin University)

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