High-Throughput Sequencing Technology and Its Applications in

Human Disease

Shuyang Deng

College of Biotechnology Science and Engineering, Beijing University of Agriculture, Chang-ping District, Hui-longguan

Town,Beijing, 102206 China

512090957@qq.com

Keywords: High-Throughput Sequencing Technology; Disease; Exome Sequencing; Chip.

Abstract: The high-throughput sequencing technology (HTS), also known as next generation sequencing, refers to the

technological advances in DNA sequencing instrumentation that enable the generation of hundreds of

thousands to millions of sequence reads per run. The advances of high-throughput, low cost and short time-

consuming democratizes HTS and paves the way for the development of a large number of novel HTS

applications in basic science as well as in translational research areas, such as clinical diagnostics,

agrigenomics, and forensic science. In recent years, HTS has been widely applied in solving biological

problems, especially in human diseases field. In this review, we provide an overview of the evolution of

HTS and discuss three important sequencing strategies HTS adopted, Roche/454, Illumina, SOLiD. We also

take the example of exome sequencing and ChIP to summarize the application of HTS in human diseases.

1 INTRODUCTION

The human genome sequence has profoundly altered

our understanding of biology, human diversity, and

disease (Reuter, 2015). The human genome project,

however, required vast amounts of time and

resources and it is clear that faster, higher

throughput, and cheaper technologies. This

stimulates the development and commercialization

of high-throughput sequencing (HTS) technologies,

as opposed to the automated Sanger method, which

is considered a first-generation technology (Van Dijk,

2014).

Compared to first-generation Sanger sequencing

technology, the second-generation sequencing

technology not only maintains high accuracy, but

also dramatically increasing sequencing speed and

reduces sequencing costs. The Human Genome

Project lasted 13 years at a cost of nearly $ 3 billion,

and just created a single human genome map. At

present, the genome sequencing using a second-

generation sequencing technology costs only a few

thousand dollars, and the cost is still decreasing. In

the world’s top several genome research centers,

hundreds of people genome sequencing in a month

has been achieved. This new generation of high-

throughput analysis allows people to study the

disease with lower cost, more comprehensively. It

breaks down the limitations of previous fluxes on

disease research, which makes it possible to expand

all-round research on disease from genomic level,

transcriptome level, Proteomics level and other

aspects, shown by Fig. 1

Fig. 1. Disease research strategies of HTS.

The research and applications of HTS in the field

of life sciences and medical are becoming more and

more widespread. The following will introduce the

important roles of HTS in disease diagnosis and

prevention from three aspects of the prenatal care,

318

Deng S.

High-Throughput Sequencing Technology and Its Applications in Human Disease.

DOI: 10.5220/0006449703180324

In ISME 2016 - Information Science and Management Engineering IV (ISME 2016), pages 318-324

ISBN: 978-989-758-208-0

tumor diagnosis and other major disease

pathogenesis.

HTS has been recognized in the field of

noninvasive prenatal care. Norton et al. (Norton,

2015) assigned pregnant women presenting for

aneuploidy screening at 10 to 14 weeks of gestation

to undergo both standard screening and cfDNA

testing. Of 18,955 women who were enrolled, results

from 15,841 were available for analysis. The AUC

for trisomy 21 was 0.999 for cfDNA testing and

0.958 for standard screening (P=0.001). Xu et al.

(Xu, 2015) carried out sequencing for single

blastomere cells and the family trio and further

developed the analysis pipeline, including recovery

of the missing alleles, removal of the majority of

errors, and phasing of the embryonic genome. The

final accuracy for homozygous and heterozygous

single-nucleotide polymorphisms reached 99.62%

and 98.39%, respectively. In addition, HTS can also

be applied to inherited heart diseases (Wilson, 2015),

cleft lip and palate (Wolf, 2015), phenylketonuria

(Gu, 2014) and so on, to prevent birth defects.

Currently, cancer is one of the major diseases

that endanger human health. As a complex disease,

its pathogenesis, typing and evolution are scientific

problems to be solved urgently. Besides, the

differences among individual cancer patients are

major challenge faced by the clinical treatment.

Using high-throughput DNA sequencing technology,

the comprehensive and systematic research on

cancer in the level of molecular biology can get a

large number of multi-dimensional tumor data set,

and extract tumor-associated genomic mutations or

modification information by scientific statistical

analysis. These will contribute to cancer prevention,

diagnosis, treatment, and overcome the road to lay

the cornerstone of the tumor for the human.

After describing the development of next-

generation sequencing in basic and clinical research,

Renkema et al. (Renkenma, 2014) suggested that

integrate data obtained using next-generation

sequencing with personalized medicine, including

use of high-throughput disease modelling as a tool to

support the clinical diagnosis of kidney diseases. To

develop an amplicon-based, next-generation

sequencing, mutation-detection assay for lung cancer

using the 454 GS Junior, Deeb et al. (Deeb, 2015)

designed fusion primers incorporating target

sequence, 454 adaptors, and multiplex identifiers to

generate 35 amplicons (median length 246 base

pairs) covering 8.9 kilobases of mutational hotspots

in AKT1, BRAF, EGFR, ERBB2, HRAS, KRAS,

NRAS, PIK3CA, and MAP2K1 genes and all exons

of the PTEN gene. In total, 25 point mutations and 4

insertions/deletions (indels) with a frequency of

5.5% to 93.1% mutant alleles were detected. Chung

et al. (Chuang, 2015) sought to investigate the

precise mutational landscape of four well-validated

Genetically engineered mouse models (GEMMs)

representing three types of cancers, non-small cell

lung cancer (NSCLC), pancreatic ductal

adenocarcinoma (PDAC) and melanoma. Thibodeau

et al. (Thibodeau, 2016) used next generation

sequencing to identify a pattern of genomic variation

associated with the development of brain metastases

in non-small cell lung cancer (NSCLC). While no

single variant was associated with brain metastasis,

this study implicated PI3K/AKT signaling and, in

particular, variants of TP53 as crucial for

determining the potential development of NSCLC

brain metastasis.

HTS has also been applied to great effect in the

field of other diseases. Information gained from

high-throughput DNA sequencing of

immunoglobulin genes (Ig-seq) can be applied to

detect B-cell malignancies with high sensitivity, to

discover antibodies specific for antigens of interest,

to guide vaccine development and to understand

autoimmunity (Georgiou, 2014). Redin et al. (Redin,

2014) reported the alternative strategy of targeted

high-throughput sequencing of 217 genes in which

mutations had been reported in patients with

intellectual disability or autism as the major clinical

concern. They analysed 106 patients with intellectual

disability of unknown aetiology following array-

CGH analysis and other genetic investigations. Saare

et al. (Saare, 2014) used a novel approach to

determine the endometriotic lesion-specific miRNAs

by high-throughput small RNA sequencing of paired

samples of peritoneal endometriotic lesions and

matched healthy surrounding tissues together with

eutopic endometria of the same patients. Results

indicated that only particular miRNAs with a

significantly higher expression in endometriotic cells

can be detected from lesion biopsies, and can serve

as diagnostic markers for endometriosis. Krauskopf

et al. (Krauskopf, 2015) focused on examining

global circulating miRNA profiles in serum samples

from subjects with liver injury caused by accidental

acetaminophen (APAP) overdose. Upon applying

next generation high-throughput sequencing of small

RNA libraries, they identified 36 miRNAs, including

3 novel miRNA-like small nuclear RNAs, which

were enriched in the serum of APAP overdosed

subjects. Comas et al. (Comas, 2016) described

current next generation sequencing approaches

applied to the Mycobacterium tuberculosis complex,

their contribution to the diagnostics and

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epidemiology of the disease and the efforts that were

being undertaken to make the technology accessible

to public health and clinical microbiology

laboratories.

2 THE FUNDAMENTAL OF HTS

A. Roche/454

In the Roche/454 approach, the library fragments are

mixed with a population of agarose beads whose

surfaces carry oligonucleotides complementary to

the 454-specific adapter sequences on the fragment

library, so each bead is associated with a single

fragment (Mardis, 2008). Water micelles that also

contain PCR reactants, and thermal cycling

(emulsion PCR) of the micelles produces

approximately one million copies of each DNA

fragment on the surface of each bead. These

amplified single molecules are then sequenced en

masse. First the beads are arrayed into a picotiter

plate (PTP; a fused silica capillary structure) which

holds a single bead in each of several hundred

thousand single wells, which provides a fixed

location at which each sequencing reaction can be

monitored. Enzymecontaining beads that catalyze

the downstream pyrosequencing reaction steps are

then added to the PTP and the mixture is centrifuged

to surround the agarose beads. The PTP is seated

opposite a CCD camera that records the light emitted

at each bead. The first four nucleotides (TCGA) on

the adapter fragment adjacent to the sequencing

primer added in library construction correspond to

the sequential flow of nucleotides into the flow cell.

This strategy allows the 454 base-calling software to

calibrate the light emitted by a single nucleotide

incorporation.

Illumina/Solexa

The Illumina system utilizes a sequencing-by-

synthesis approach in which all four nucleotides are

added simultaneously to the flow cell channels,

along with DNA polymerase, for incorporation into

the oligo-primed cluster fragments. Specifically, the

nucleotides carry a base-unique fluorescent label and

the 3’-OH group is chemically blocked such that

each incorporation is a unique event. An imaging

step follows each base incorporation step, during

which each flow cell lane is imaged in three 100-tile

segments by the instrument optics at a cluster density

per tile of 30,000. After each imaging step, the 3’

blocking group is chemically removed to prepare

each strand for the next incorporation by DNA

polymerase (Mardis, 2008).

Solid

SOLiD systems isolate and amplify single DNA

molecules to construct a library for sequencing by a

process known as emulsion PCR (Tawfik, 1998).

Emulsification of an oil-water interface leads to the

formation of droplets, with each droplet, referred to

as a microreactor, containing a bead that is

covalently bound to a single DNA template. PCR

amplification is then performed across the surface of

the bead to generate clonally amplified fragments.

For SOLiD, after emulsion PCR, the 3’ ends of the

DNA template on the bead are modified to permit

chemical linkage to the surface of a glass slide.

When sequencing reagents containing DNA ligase

are flowed over the slide, a fluorescent signal is

generated that is captured by a CCD camera for base

calling. SOLiD sequencing is classified as

sequencing-by-ligation, because sequencing is

determined according to the selective mismatch

sensitivity of DNA ligase to fluorescently labeled

probes.

Evolution of HTS Platform

Comparing with 454, Illumina and SOLiD

sequencing are more suitable, because of their higher

throughput. For this reason, transcriptome profiling

and ChIP-seq studies have mostly used Illumina or

SOLiD sequencing (Wang, 2009; Park, 2009). By

contrast, the reads generated by these technologies

are initially too short for de novo genome

assemblies. Thus, 454 is the preferred technology for

this type of application and enables exciting

discoveries such as the first million bp of a

Neandertal genome (Green, 2006). Another

important field of 454 is metagenomics, for example

uncovering the potential cause of the disappearance

of the honeybee (Cox-Foster, 2007).

Fig. 1. Maximum read length HTS platform.

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Fig. 2. Maximum throughput HTS platform.

With the rapid improvement of sequencing

machines and chemistries, Illumina technology can

now generate reads of several hundreds of bp long,

shown by Fig. 2. Thus, although reads produced by

Illumina still shorter than that produced by 454, de

novo genome assembly and metagenomics can now

also be performed with Illumina sequencing. Illumina

has been achieved remarkable increase in throughput,

which currently offers the highest throughput per run

with the lowest per base cost (Liu, 2012), shown by

Fig. 3. A summary of the advantages and drawbacks

of the different HTS systems is presented in Table 1.

3 THE APPLICATION OF HTS IN

HUMAN DISEASE RESEARCH

HTS is a new molecular detection technology with

high speed, high accuracy and high throughput. Its

research and application become more and more

widely in the field of life sciences and medical.

Genome data obtained by analysis and mining, and

genetic variation of information extracted from the

disease can provide a scientific basis for disease

diagnosis, treatment and prevention (Zaghloul,

2010).

In this part, the application of new generation

HTS in human disease is reviewed with exome

sequencing and Chip sequencing technology as an

example.

Application of Exome Sequencing

in Human Diseases

Exome sequencing is a high-throughput sequencing

method by using special means to enrich the whole

exome. The basic processes include enrichment of

exome region sequences, high throughput

sequencing and bioinformatics analysis of

sequencing data.

The exon region contains the information

required by the synthesis of proteins, and covers

most of the functional variants associated with the

phenotype of the individual. Howerver, these exon

regions of the encoded protein account for only

about 1% of the human genome, thus it can greatly

improve the efficiency of exon region research

compared to the conventional PCR methods. In the

case of relatively high cost of sequencing, we can

obtain data with deeper coverage and higher

accuracy of sequencing coverage, and more coding

region information of the individual using the exome

sequencing under the same cost.

TABLE I. Pros and cons of the different HTS

Technology Pros Cons

Roche/454

The long reads (1 kb maximum) are easier to

map to a reference genome, and are an

advantage for de novo genome assemblies or

for Metagenomics applications. Run times

are relatively fast (~23 h)

Relatively low throughput (about 1 million reads,

700 Mb sequence data) and high reagent cost.

High error rates in homopolymer repeats .

Illumine/Solexa

Illumina is currently the leader in the HTS

industry and most library preparation

protocols are compatible with the Illumina

system. In addition, Illumina offers the

highest throughput of all platforms and the

lowest per-base cost. Read lengths of up to

300 bp, compatible with almost all types of

application.

Sample loading is technically challenging; owing

to the random scattering of clusters across the

flow cells library concentration must be tightly

controlled. Overloading results in overlapping

clusters and poor sequence quality.

SOLID

Second highest throughput system on the

market. The SOLiD system is widely

claimed to have lower error rates, 99.94%

accuracy, than most other systems owing to

the fact that each base is read twice.

Shortest reads (75 nt maximum) of all platforms,

and relatively long run times. Less-well-suited for

de novo genome assembly. The SOLiD system is

much less widely used than the Illumina system

and the panel of sample preparation kits and

services is less well developed.

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Exome sequencing is an effective method to

detect pathogenicity genes and susceptibility loci at

the genomic level. It can not only rapidly locate the

gene of single gene disease, but also be used to study

the common diseases caused by mutations, such as

diabetes, hypertension and tumor.

Nat Genet et al. (Ng, 2009) reported on the

targeted capture and massively parallel sequencing

of the exomes of 12 humans. Using FSS as a proof-

of-concept, they showed that candidate genes for

Mendelian disorders could be identified by exome

sequencing of a small number of unrelated, affected

individuals. In the same year, Nat Genet et al. (Ng,

2010) demonstrated the first successful application

of exome sequencing to discover the gene for a rare

mendelian disorder of unknown cause. Jones et al.

(Jones, 2010) determined the exomic sequences of

eight tumors after immunoaffinity purification of

cancer cells. Through comparative analyses of

normal cells from the same patients, they identified

four genes that were mutated in at least two tumors,

PIK3CA, KRAS, PPP2R1A and ARID1A. The

nature and pattern of the mutations suggested that

PPP2R1A functions as an oncogene and ARID1A as

a tumor-suppressor gene. Harbour et al. (Harbour,

2010) used exome capture coupled with massively

parallel sequencing to search for metastasis-related

mutations in highly metastatic uveal melanomas of

the eye. Their findings implicated loss of BAP1 in

uveal melanoma metastasis and suggested that the

BAP1 pathway might be a valuable therapeutic

target. Seshagiri et al. (Seshagiri, 2012) analysed

systematically more than 70 pairs of primary human

colon tumours by applying next-generation

sequencing to characterize their exomes,

transcriptomes and copy-number alterations. Their

analysis for significantly mutated cancer genes

identified 23 candidates, including the cell cycle

checkpoint kinase ATM. Li et al. (Li, 2011)

discovered novel inactivating mutations of ARID2 in

four major subtypes of HCC. Notably, 18.2% of

individuals with HCV-associated HCC in the United

States and Europe harbored ARID2 inactivation

mutations, suggesting that ARID2 was a tumor

suppressor gene that was relatively commonly

mutated in this tumor subtype. Xu et al. (Xu, 2011)

examined the possibility that rare de novo protein-

altering mutations contribute to the genetic

component of schizophrenia by sequencing the

exomes of 53 sporadic cases, 22 unaffected controls

and their parents. They analyses suggested a major

role for de novo mutations in schizophrenia as well

as a large mutational target, which together provide a

plausible explanation for the high global incidence

and persistence of the disease. Glessner et al.

(Glessner, 2014) studied 538 CHD trios using

genome-wide dense single nucleotide polymorphism

arrays and whole exome sequencing. Integrating de

novo variants in whole exome sequencing and CNV

data suggested that ETS1 was the pathogenic gene

altered by 11q24.2-q25 deletions in Jacobsen

syndrome and that CTBP2 was the pathogenic gene

in 10q subtelomeric deletions.

ChIP and its Application in Human

Disease

ChIP uses an immune reagent specific for a DNA

binding factor to enrich target DNA sites to which

the factor is bound in the living cell. The enriched

DNA sites are then identified and quantified.

Because of the gigabase-size genomes of vertebrates,

ChIP cannot combine high accuracy, whole-genome

completeness, and high binding-site resolution.

Chromatin immunoprecipitation followed by

sequencing, short for ChIPSeq, is a high-throughput

method combining ChIP with a HTS (Johnson,

2007). The ChIPSeq differs from other large-scale

ChIP methods such as ChIPchip in design, data

produced, and cost. ChIP-seq has the advantage of

high-resolution, low-noise, high coverage to study

the protein-DNA interactions (Schones, 2008).

ChIP-seq can be applied to any species with known

genome sequence, and study the interaction between

any kind of DNA-related protein and its Target

DNA.

ChIPSeq illustrates the power of new sequencing

platforms, such as those from Solexa/ Illumina and

454, to perform sequence census counting assays.

The generic task in these applications is to identify

and quantify the molecular contents of a nucleic acid

sample whose genome of origin has been sequenced

(Johnson, 2007).

With the reduction of sequencing costs, ChIP-seq

gradually becomes a common method of studying

gene regulation and epigenetic mechanism. The

applications of ChIP-seq in human disease research

are becoming more and more extensive.

Robertson et al. (Robertson, 2007) used ChIP-seq

to map STAT1 targets in interferon-bold gamma

(IFN-bold gamma)-stimulated and unstimulated

human HeLa S3 cells, and compared the method’s

performance to ChIP-PCR and to ChIP-chip for four

chromosomes. By ChIP-seq, using 15.1 and 12.9

million uniquely mapped sequence reads, and an

estimated false discovery rate of less than 0.001,

they identified 41,582 and 11,004 putative STAT1-

binding regions in stimulated and unstimulated cells,

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respectively. Lin et al. (Lin, 2009) applied

expression profiling to identify the response program

of PC3 cells expressing the AR (PC3-AR) under

different growth conditions (i.e. with or without

androgens and at different concentration of

androgens) and then applied the newly developed

ChIP-seq technology to identify the AR binding

regions in the PC3 cancer genome. They found that

the comparison of MOCK-transfected PC3 cells with

AR-transfected cells identified 3,452 differentially

expressed genes (two fold cutoff) even without the

addition of androgens (i.e. in ethanol control),

suggesting that a ligand independent activation or

extremely low-level androgen activation of the AR.

ChIP-Seq analysis revealed 6,629 AR binding

regions in the cancer genome of PC3 cells with an

FDR (false discovery rate) cut off of 0.05. Hurtado

et al. (Hurtado, 2011) used ChIP-seq to research

breast cancers and found that FOXA1 was a key

determinant that can influence differential

interactions between estrogen receptor-α (ER) and

chromatin. Ross-Innes et al. (Ross-Innes, 2012)

mapped genome-wide ER-binding events, by

chromatin immunoprecipitation followed by ChIP-

seq, in primary breast cancers from patients with

different clinical outcomes and in distant ER-

positive metastases. Results showed that there was

plasticity in ER-binding capacity, with distinct

combinations of cis-regulatory elements linked with

the different clinical outcomes. Wang et al. (Wang,

2012) analyzed genome-wide occupancy patterns of

CTCF by ChIP-seq in 19 diverse human cell types,

including normal primary cells and immortal lines.

Results revealed a tight linkage between DNA

methylation and the global occupancy patterns of a

major sequence-specific regulatory factor. Lee et al.

(Lee, 2012) introduced genome-wide studies that

mappped the binding sites of CTCF and its

interacting partner, cohesin, using chromatin

immunoprecipitation coupled with ChIP-seq

revealded that CTCF globally co-localizes with

cohesin.

4 CONCLUSIONS

Ongoing cost reduction and the development of

standardized pipelines will probably make HTS a

standard tool for a multifaceted approach involving

clinical and research laboratories, bioinformatics

scientists, biotechnology companies, and regulatory

agencies in the near future.

Nevertheless, the implementation of HTS faces

significant challenges, in particular high data storage

and complex processing. With time goes on, the

amount of human genomes will be far more than the

already impressive amount of sequence data

available. Due to so many people’s genomes

sequenced, it is in great need to increase the data

storage capacity, speed up the establishment and

maintenance of databases, and develop efficient data

retrieval methods. For complex diseases, the

relationship between the massive sequencing

genome data and disease is not clear. Disease

pathogenic and development process cannot be

guaranteed only by obtained genomic information.

Biochemical data, such as transcriptome, proteome,

macro-genome, etc. as well as CT and MRI images

are needed to combine together to construct a large-

scale multi-dimensional life health data collection.

With the rapid development of cloud computing

technology, the storage, analysis and monitoring of

various vital signs and data will gradually be

realized. Accurate analysis and data mining will

decipher the causes of human disease and promote

the development of precision medicine.

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