An Overview of Next-Generation

Sequencing
Article Published: March 17, 2021|Athina Gkazi, PhD

Over the last 56 years, researchers have been developing methods and
technologies to assist in the determination of nucleic acid sequences in biological
samples. Our ability to sequence DNA and RNA accurately has had a great impact
in numerous research fields. This article discusses what next-generation
sequencing (NGS) is, advances in the technology and its applications.

Contents

What is next-generation sequencing?

 Next-generation sequencing methods

- Sequencing platforms/ sequencing technology

- Main steps of 2G sequencing methods and next-generation sequencing
library prep
Short-read vs long-read sequencing
What's the difference between whole-exome vs whole-genome sequencing?
Next-generation sequencing data analysis
Next-generation sequencing bottlenecks
Next-generation sequencing applications
Next-generation sequencing key terms and abbreviations

What is next-generation sequencing?

The structure of DNA was determined in 1953 by Watson and Crick based on the
fundamental DNA crystallography and X-ray diffraction work of Rosalind
Franklin.1,2 However, the first molecule to be sequenced was actually RNA – tRNA –
in 1965 by Robert Holley and RNA of bacteriophage MS2 later on.3,4 Various
research groups then began adapting these methods to sequence DNA with a
breakthrough coming in 1977 by Fredrick Sanger and colleagues, developing the
chain-termination method.5 By 1986, the first automated DNA sequencing method
had been developed.6,7 This was the beginning of a golden era for the
development and refinement of sequencing platforms, including the pivotal
capillary DNA sequencer.

The chain-termination method, also known as Sanger sequencing, uses a DNA

sequence of interest as a template for a PCR that adds modified nucleotides, called
dideoxyribonucleotides (ddNTPs), to the DNA strand during the extension step.8
When the DNA polymerase incorporates a ddNTP, the extension ceases leading to
the generation of numerous copies of the DNA sequence of all lengths spanning
the amplified fragment. These chain-terminated oligonucleotides are then size
separated using gel electrophoresis in early methods, or capillary tubes in later
automated capillary sequencers and the DNA sequence is determined. With these
immense technological advances, the human genome project was completed in
2003.9 In 2005, the first commercially available NGS platform, or second
generation (2G) as it has become, was introduced, able to amplify millions of
copies of a particular DNA fragment in a massively paralleled way in contrast to
Sanger sequencing.10

The key principles behind Sanger sequencing and 2G NGS share some
similarities.11,12 In 2G NGS, the genetic material (DNA or RNA) is fragmented, to
which oligonucleotides of known sequences are attached, through a step known as
adapter ligation, enabling the fragments to interact with the chosen sequencing
system. The bases of each fragment are then identified by their emitted signals.
The main difference between Sanger sequencing and 2G NGS stems from
sequencing volume, with NGS allowing the processing of millions of reactions in
parallel, resulting in high-throughput, higher sensitivity, speed and reduced cost. A
plethora of genome sequencing projects that took many years with Sanger
sequencing methods could now be completed within hours using NGS.

There are two main approaches in NGS technology, short-read and long-read
sequencing, each with its own advantages and limitations (Table 1).13 The main
scope for investing in the development of NGS is its wide applicability in both
clinical and research settings. In clinical settings, NGS is used to diagnose various
disorders, via identification of germline or somatic mutations.14,15 The shift
towards NGS in clinical practice is justified by the power of the technique paired
with the continually declining costs. NGS is also a valuable tool in metagenomic
studies and used for infectious disease diagnostics, monitoring and
management.16,17 In 2020, NGS methods were pivotal in characterizing the SARS-
CoV-2 genome and are constantly contributing in monitoring the COVID-19
pandemic.18,19
Figure 1: The evolution of sequencing methodologies.

Next-generation sequencing methods

The term NGS is often taken to mean 2G technologies, however, third (3G) and
fourth (4G) generation technologies have since evolved that work on different
underlying principles.

Sequencing platforms/ sequencing technology

Second-generation sequencing methods are well-established and share many

features in common. They can, however, be subdivided according to their
underlying detection chemistries including sequencing by ligation (incorporating
nanoball) and sequencing by synthesis (SBS), which further divides into proton
detection, pyrosequencing and reversible terminator (Figure 2).
Figure 2: Diagram representing the principle 2G sequencing platforms and
chemistries.

Proton detection sequencing relies on counting hydrogen ions released during the
polymerization of DNA. Unlike other techniques, it does not use fluorescence and
does not use modified nucleotides or optics. Instead, pH changes are detected by
semiconductor sensor chips and converted to digital information.20

Pyrosequencing utilizes the detection of pyrophosphate generation and light

release to determine whether a specific base has been incorporated in a DNA
chain.21,22

By far the most popular SBS method is reversible terminator sequencing which
utilizes ‘’bridge-amplification’’. During the synthesis reactions, the fragments bind
to oligonucleotides on the flow cell, creating a bridge from one side of the
sequence (P5 oligo on flow cell) to the other (P7), which is then amplified. The
added fluorescently-labeled nucleotides are detected using direct imaging.23
Unlike SBS, sequencing by ligation does not use DNA polymerase to create a
second strand. The sensitivity of DNA ligase to base-pairing mismatches is utilized
instead, with the fluorescence produced used to determine the target sequence.
Digital images taken after each reaction are then used for analysis. DNA nanoball
sequencing is a form of sequencing by ligation that exploits rolling circle
replication. Concatenated DNA copies are compacted into DNA nanoballs and
bound to sequencing slides in a dense grid of spots ready for ligation-based
sequencing reactions.24,25 Whilst the nanoball technique reduces running costs,
the short sequences produced can be problematic for read mapping.

Continue reading below...

How To Guide

How To Optimize Your NGS Workflow

Read More

2G NGS technologies in general offer several advantages over alternative

sequencing techniques, including the ability to generate sequencing reads in a fast,
sensitive and cost-effective manner. However, there are also disadvantages,
including poor interpretation of homopolymers and incorporation of incorrect
dNTPs by polymerases, resulting in sequencing errors. The short read lengths also
create the need for deeper sequencing coverage to enable accurate contig and
final genome assembly.26–30 The major disadvantage of all 2G NGS techniques is
the need for PCR amplification prior to sequencing. This is associated with PCR bias
during library preparation (sequence GC-content, fragment length and false
diversity) and analysis (base errors/favoring certain sequences over others).

The introduction of 3G sequencing circumvents the need for PCR, sequencing

single molecules without prior amplification steps. The first single molecule
sequencing (SMS) technology was developed by Stephen Quake and colleagues.31
Here, sequence information is obtained with the use of DNA polymerase by
monitoring the incorporation of fluorescently labeled nucleotides to DNA strands
with single base resolution. Depending on the method and the instrument used,
some of the advantages of 3G NGS include:

• Real-time monitoring of nucleotide incorporation

• Non-biased sequencing and
• Longer read lengths

Nevertheless, high costs, high error rates, large quantities of sequencing data and
low read depth can be problematic.32,33

In 4G systems the single-molecule sequencing of 3G is combined with nanopore

technology. Similar to 3G, nanopore technology requires no amplification and uses
the concept of single molecule sequencing but with the integration of tiny biopores
of nanoscale diameter (nanopores) through which the single molecule passes and
is identified. The 4G systems currently offer the fastest whole genome sequence
scan but are still quite expensive, error prone compared to 2G techniques and
relatively new. Consequently, there is currently less extensive data available for the
technique.34

Main steps of 2G sequencing methods and next-generation sequencing

library prep
Regardless of the 2G NGS method chosen, there are several main steps that must
be tailored and optimized to the target (RNA or DNA) and sequencing system
selected.

(1) Sample preparation (pre-processing)

Nucleic acids (DNA or RNA) are extracted from the selected samples (blood,
sputum, bone marrow etc.). Extracted samples are quality control (QC) checked,
using standard methods (spectrophotometric, fluorometric or gel electrophoretic).
If using RNA, this must be reverse transcribed into cDNA, however some library
preparation kits may include this step.

(2) Library preparation

Random fragmentation of the cDNA or DNA, typically by enzymatic treatment or

sonication, is performed. The optimal fragment length depends on the platform
being used. It may be necessary to run a small amount of fragmented sample on
an electrophoresis gel when optimizing this process. These fragments are then
end-repaired and ligated to smaller generic DNA fragments called adapters.
Adapters have defined lengths with known oligomer sequences to be compatible
with the applied sequencing platform and identifiable where multiplex sequencing
is performed. Multiplex sequencing, using individual adapter sequences per
sample, enables large numbers of libraries to be pooled and sequenced
simultaneously in a single run. This pool of DNA fragments with adapters attached
are known as a sequencing library.

Size selection may then be performed, by gel electrophoresis or using magnetic

beads, to remove any fragments that are too short or too long for optimal
performance on the sequencing platform and protocol selected. Library
enrichment/amplification is then achieved using PCR. In techniques involving
emulsion PCR, each fragment is bound to a single emulsion bead which will go on
to form the basis of sequencing clusters. Amplification is often followed by a
“clean-up” step (e.g., using magnetic beads) to remove undesired fragments and
improve sequencing efficiency.

The final libraries can undergo QC checks using qPCR, to confirm DNA quality and
quantity. This will also allow the correct concentration of sample to be prepared
for sequencing.

(3) Sequencing

Depending on the selected platform and chemistry, clonal amplification of library

fragments may occur prior to sequencer loading (emulsion PCR) or on the
sequencer itself (bridge PCR). Sequences are then detected and reported according
to the platform selected.35

(4) Data analysis

The generated data files are analyzed depending on the workflow used. Analysis
methods are highly dependent on the aim of the study.36–38

Whilst they may reduce the amount of samples that can be analyzed in a given run,
paired-end and mate pair sequencing offer advantages in downstream data
analysis, particularly for de novo assemblies. The techniques link sequencing reads
together that are read from both ends of a fragment (paired-end) or are separated
by an intervening DNA region (mate pair).

There are clearly many options when it comes to selecting a sequencing strategy.
The following are some of the key considerations when deciding on the
appropriate library preparation and sequencing platform:

(a) Research question being asked

(b) Sample type

(c) Short-read or long-read sequencing

(d) DNA or RNA sequencing – do you need to look at the genome or transcriptome?

(e) Is the whole genome required or only specific regions?

(f) Read depth (coverage) needed – experiment-specific

(g) Extraction method

(h) Sample concentration

(i) Single end, paired end or mate pair reads

(j) Specific read length required

(K) Could samples be multiplexed?

(l) Bioinformatic tools – experiment dependent. Depending on the sample and the
biological question, the entire process of sequence analysis can be adapted.

Short-read vs long-read sequencing

The advantages and disadvantages of short- and long-read sequencing are

summarized in Table 1.

Table 1: A table of advantages and disadvantages for short vs long read

sequencing.

Advantages Limitations
Shor
· Not able to resolve structural
t-
· Higher sequence fidelity variants, phasing alleles or distinguish
read
· Cheap highly homologous genomic regions
sequ
· Can sequence fragmented DNA · Unable to provide coverage of some
enci
repetitive regions
ng

· Able to sequence genetic

regions that are difficult to
characterize with short-read seq
due to repeat sequences
Long · Lower per read accuracy
· Able to resolve structural
-read · Bioinformatic challenges, caused by
rearrangements or homologous
sequ coverage biases, high error rates in
regions
enci base allocation, scalability and limited
· Able to read through an entire
ng availability of appropriate pipelines
RNA transcript to determine the
specific isoform

· Assists de novo genome

assembly

What's the difference between whole-exome vs whole-

genome sequencing?
Whole genome sequencing (WGS) is the most widely used form of NGS and refers
to the analysis of the entire nucleotide sequence of a genome. Whole exome
sequencing (WES) on the other hand is a form of targeted sequencing that only
addresses the protein coding exons. In humans, this accounts for only around 2%
of the genome and consequently offers an opportunity for a greater depth of study
in these regions. Due to the reduced sequencing burden, WES can also offer a
more cost-effective option than WGS and reduce the volume and complexity of the
resultant sequencing data. However, by sequencing only a fraction of the genome,
vital information may be missed and the opportunity for novel discoveries is
reduced. Despite the increased, although rapidly declining, costs and the
associated data analysis challenges, WGS therefore offers a more powerful
analysis that can reveal a more complete picture.

Next-generation sequencing data analysis

Any kind of NGS technology generates a significant amount of output data. The
basics of sequence analysis follow a centralized workflow which includes a raw
read QC step, pre-processing and mapping, followed by post-alignment processing,
variant annotation, variant calling and visualization.

Assessment of the raw sequencing data is imperative to determine their quality

and pave the way for all downstream analyses. It can provide a general view on the
number and length of reads, any contaminating sequences, or any reads with low
coverage. One of the most well-established applications for computing quality
control statistics of sequencing reads is FastQC. However, for further pre-
processing, such as read filtering and trimming, additional tools are needed.
Trimming bases towards the ends of reads and removing leftover adapter
sequences generally improves data quality. More recently, ultra-fast tools have
been introduced, such as fastp, that can perform quality control, read filtering and
base correction on sequencing data, combining most features from the traditional
applications while also running two to five times faster than any of them alone.39

After the quality of the reads has been checked and pre-processing performed, the
next step will depend on the existence of a reference genome. In the case of a de
novo genome assembly, the generated sequences are aligned into contigs using
their overlapping regions. This is often done with the assistance of processing
pipelines that can include scaffolding steps to help with contig ordering,
orientation and the removal of repetitive regions, thus increasing the assembly
continuity.40,41 If the generated sequences are mapped (aligned) to a reference
genome or transcriptome, variations compared to the reference sequence can be
identified. Today, there is a plethora of mapping tools (more than 60), that have
been adapted to handle the growing quantities of data generated by NGS, exploit
technological advancements and tackle protocol developments.42 One difficulty,
due to the increasing number of mappers, is being able to find the most suitable
one. Information is usually scattered through publications, source codes (when
available), manuals and other documentation. Some of the tools will also offer a
mapping quality check that is necessary as some biases will only show after the
mapping step. Similar to quality control prior to mapping, the correct processing of
mapped reads is a crucial step, during which duplicated mapped reads (including
but not limited to PCR artifacts) are removed. This is a standardized method, and
most tools share common features. Once the reads have been mapped and
processed, they need to be analyzed in an experiment-specific fashion, what is
known as variant analysis. This step can identify single nucleotide polymorphisms
(SNPs), indels (an insertion or deletion of bases), inversions, haplotypes,
differential gene transcription in the case of RNA-seq and much more. Despite the
multitude of tools for genome assembly, alignment and analysis, there is a
constant need for new and improved versions to ensure that the sensitivity,
accuracy and resolution can match the rapidly advancing NGS techniques.

The final step is visualization, for which data complexity can pose a significant
challenge. Depending on the experiment and the research questions posed, there
are a number of tools that can be used. If a reference genomes is available , the
Integrated Genome Viewer (IGV)is a popular choice43, as is the Genome Browser. If
experiments include WGS or WES, the Variant Explorer is a particularly good tool as
it can be used to sieve through thousands of variants and allow users to focus on
their most important findings. Visualization tools like VISTA allow for comparison
between different genomic sequences. Programs suitable for de novo genome
assemblies44 are more limited. However, tools like Bandage and Icarus have been
used to explore and analyze the assembled genomes.

Next-generation sequencing bottlenecks

NGS has enabled us to discover and study genomes in ways that were never
possible before. However, the complexity of the sample processing for NGS has
exposed bottlenecks in managing, analyzing and storing the datasets. One of the
main challenges is the computational resources required for the assembly,
annotation, and analysis of sequencing data.45 The vast amount of data generated
by NGS analysis is another critical challenge. Data centers are reaching high
storage capacity levels and are constantly trying to cope with increasing demands,
running the risk of permanent data loss.46 More strategies are continuously being
suggested with the aim to increase efficiency, reduce sequencing error, maximize
reproducibility and ensure correct data management.

Next-generation sequencing applications

Since the early 2000s NGS has become an invaluable tool in both research and
clinical/diagnostic settings for modern medicine and in drug discovery, with the
use of methods including WGS, WES, targeted sequencing, transcriptome,
epigenome and metagenome sequencing dramatically increasing. Figure 3
summarizes workflows and options for targeting different datasets.

Figure 3: Flow diagram indicating possible sequencing strategies for different

sample types.

Through WGS, researchers are able to study not only genes and their involvement
in disease in humans and animals, but also characteristics of microbial and
agricultural populations, providing important epidemiological and evolutionary
data.47–52 Thus far, there has been a plethora of studies where mutations,
rearrangements and fusion events were identified using WGS. Currently, WGS is
used for the surveillance of antimicrobial resistance, one of the major global health
challenges.53,54 As the costs are constantly decreasing, WGS is more frequently
used for resequencing the entire human genome in clinical samples and may soon
become routine in clinical practice.55 Ultimately, WGS will be needed to assign
functionality to the remaining majority of the genome and decipher its role in
diseases.

Their more focused nature make WES and targeted sequencing attractive options
for population and clinical studies.56,57 Despite having more limitations as the
name suggests, WES is an important clinical tool in the personalized medicine field.
Genetic diagnoses for certain diseases, like cancer, as well as genetic
characterization for other disorders can be achieved with this method in a more
cost-effective way than WGS.

In addition to the many applications that NGS has in sequencing DNA, it can also
be used for RNA analysis. This enables, for example, the genomes of RNA viruses,
such as SARS and influenza, to be determined. Importantly, RNA-seq is frequently
used in quantitative studies, facilitating not only the identification of transcribed
genes in a DNA genome, but also the level at which they are transcribed
(transcription level) according to the relative abundance of RNA transcripts.
Potential rearrangements of the DNA sequences may also be identified through
the identification of novel transcripts.58,59

Epigenomic sequencing allows the study of changes caused by histone

modifications and DNA methylation. There are different methods employed for the
study of epigenetic mechanisms, including whole genome bisulfate sequencing
(WGBS), chromatin immunoprecipitation (ChIP-seq) and methylation dependent
immunoprecipitation (MeDIP-seq) followed by sequencing.60,61 Depending on the
selected method, the complete DNA methylome and histone modification profiles
can be mapped and studied, gaining insights into genomic regulatory mechanisms.

Metagenomic sequencing can provide information for samples collected in a

specific environment. It enables the comparison of differences and interactions
between mixed microbial populations, as well as host responses. Some of the
potential applications of metagenomic sequencing include, but are not limited to,
infectious disease diagnostics and infection surveillance, antimicrobial resistance
monitoring, microbiome studies and pathogen discovery.62

Technological advances in sample preparation, sequencing technologies and data

analysis mean that NGS is also being used at the single cell level to study
heterogeneities and rare changes in DNA, RNA and the epigenome.

Next-generation sequencing key terms and abbreviations

Table 2: Key terms and abbreviations relating to NGS.

DNA Deoxyribonucleic acid

RNA Ribonucleic acid

tRNA Transfer ribonucleic acid

NGS Next-generation sequencing

PCR Polymerase chain reaction

cDNA Complementary DNA

gDNA Genomic DNA

RNA-seq RNA-sequencing

SMS Single molecule sequencing

SBS Sequencing by synthesis

WGS Whole genome sequencing

WES Whole exome sequencing

WGBS Whole genome bisulfate sequencing

ChIP-seq Chromatin immunoprecipitation sequencing

Methylation dependent immunoprecipitation followed by

MeDIP-seq sequencing

P5 Primer 5 (sequencing adapter)

P7 Primer 7 (sequencing adapter)

3G Third-generation sequencing

4G Fourth-generation sequencing

dNTPs Deoxynucleoside triphosphate

FastQC Fast quality control

Flow cell Glass slide containing fluidic channels

Library Pool of DNA fragments with adapters attached

Indel Insertion or deletion of bases

Adapters Platform-specific sequences for fragment recognition

fastp Fast preprocessor

De novo Novel genome sequencing in the absence of a reference

sequencing sequence

Contigs From “contiguous” - overlapping DNA fragments

SNP Single nucleotide polymorphism

Created by linking contigs together using additional

Scaffold information

SBL Sequencing by ligation

Reading a sequencing fragment from both ends and linking

Paired-end the data

Linking sequencing reads separated by an intervening DNA

Mate pair region

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