Assignment

Applied Genomics and

Proteomics

MTECH (BIOTECHNOLOGY) 3rd SEM

2014-2016

Topic: Applications of NGS in Research

SUBMITTED TO: Dr. Vipin Singh

Dr. Chanderdeep Tandon
Dr. Archana Chaturvedi
Amity Institute of Biotechnology

SUBMITTED BY:RENU RAWAT

Enroll: A0510714028
Roll No.: 1027

INTRODUCTION
DNA sequencing is the process by which we extract the sequence of A, C, T, and G of the entire
DNA. Since the introduction of the Sanger sequencing method in the 1970s

[1]

, DNA sequencing

technology has enabled enormous advances in molecular biology and genetics. This technology has been
used for many projects, such as the Human Genome Project, Rice Genome Project and Swine Genome
Project, as well as genome projects of many other species.
However, there were a few disadvantages of this sequencing technology. These include its low
throughput, high cost and operation difficulties. Due to these disadvantages Sanger sequencing have
limited its use in deeper and more complex genome analyses [2]. The recent introduction of nextgeneration sequencing (NGS) technology, with its high-throughput capacity and low cost, has largely
overcome these problems, and these technologies have been applied in various fields of life sciences,
including forensics, disease diagnosis, agrigenomics and ancient DNA analysis. In this article, the use of
NGS technology in forensic science is reviewed with the aim of providing a reference for future frontier
research and application in forensic science.

APPLICATIONS
1. IN CANCER RESEARCH [3]
With the development and improvement of new sequencing technology, next generation
sequencing (NGS) has been applied increasingly in cancer genomics research over the past
decades. NGS is used to identify novel and rare cancer mutations, detect familial cancer mutation
carriers, and provide molecular rationale for appropriate targeted therapy.
Identification of novel cancer mutations using NGS
NGS technologies have enabled efficient and accurate detection of novel and rare somatic
mutations. NGS has been successfully employed to identify novel mutations in a variety of
cancers, including bladder cancer, renal cell carcinoma, small cell lung cancer, prostate cancer,
acute myelogenous leukemia, and chronic lymphocytic leukemia. Whole genome sequencing
with NGS was used in patients with a rare form of acute promyelocytic leukemia and
successfully identified a novel PML-RARA genetic recombination that was undetectable with
standard cytogenetic techniques. Gui et al sequenced the exomes of 9 transitional cell carcinoma
tumors to find somatic mutations, then screened in tumor samples from 88 individuals with
transitional cell carcinoma at different stages and grades. They found 55 notable mutations
related to transitional cell carcinoma, 49 of which were first found in bladder cancer.
Furthermore, they sequenced the whole exomes of 10 clear cell renal cell carcinomas and
screened thousands of genes in an additional 88 samples, ultimately discovering 12 new mutated
genes. Both studies were published in Nature Genetics in the same year.
In another study, Keller et al. used specific target selection and NGS to identify novel
SNPs in genes already associated with glioblastoma. Over 6000 SNPs, including more than 1300
SNPs located in targeted genes, were identified.
By using NGS, numerous novel genetic aberrations and associated potential therapeutic
targets have been found in many cancers. Many of these studies on new genetic aberrations were
summarized by Tran et al.

NGS in hereditary cancer syndrome genetic testing

About 5% 10% of cancers is hereditary. Genetic testing has been used for hereditary
cancer patients for more than ten years in the US and Europe. The development of NGS provided
many opportunities for genetic testing. Walsh et al. used target region capture and NGS to detect
21 genes associated with hereditary breast and ovarian cancer. This combined method allowed
detection of several kinds of variations, including single nucleotide substitutions (SNPs), small
insertions and deletions, and large genomic duplications and deletions.
NGS provides a good solution for detecting rare variations. Because it allows testing of
multiple genes at once, NGS greatly improves the variation detection rate. Many patients with
hereditary cancer have tested negative for genetic variations, but with NGS, it is easier to find
causative mutations. In a study of 300 high-risk breast cancer families, Walsh et al. found
previously undetected mutations in 52 probands. Ozcelik et al. introduced a method that used
long-range PCR plus NGS to detect BRCA1 and BRCA2 and demonstrated that it was useful for
BRCA testing. A similar method has also been reported previously by Hernan et al. and De
Leeneer et al.
NGS for personalized cancer treatment
NGS is also used to improve rationally designed individualized medicine. For example,
NGS has been used in the treatment of pancreatic cancer. It has been also used in the detection of
epidermal growth factor receptor (EGFR) deletions in non-small cell lung cancer, which showed
important pathogenetic and clinical implications for patients with nonsmall cell lung cancer. In
addition, it has been used in the detection of PML-RARA fusion gene in acute promyelocytic
leukemia, which led to a change of a patients therapeutic schedule.
In an inspiring study, genetics researchers at Washington University did whole genome
and transcriptome sequencing for a researcher in their team, who had adult acute lymphoblastic
leukemia. The cancer relapsed twice in 10 years from the time of first diagnosis. Then, his
colleagues found a clue about the disease through RNA sequencing. Their results showed that a
normal gene, FLT3, was wildly active in the leukemia cells. Luckily, however, the drug sunitinib,
which is approved to treat advanced renal cancer, inhibits FLT3. With sunitinib treatment, his
blood counts appeared more normal. This is a very successful case of translating NGS into
clinical practice.

Table1: Number of cancer genomes sequenced [4]

Detection of circulating cancer DNA by NGS

Rare mutations in circulating DNA have long been used to detect somatic mutation for
cancer diagnosis and management. It is difficult to identify rare mutations in tumor suppressor
genes like TP53, which is highly mutated throughout the gene. NGS proved to be the costeffective method to detect and measure the allele frequency of TP53 and other tumor gene
mutations in the plasma. Forshew et al. developed a tagged amplicon deep sequencing (TAmSeq) method that used NGS and designed primers to amplify approximately 6000 bases that
covered the selected regions of cancer related genes, including EGFR, TP53 and KRAS. By
using plasma samples, they showed that the method could identify mutations in TP53 at allelic
frequencies of 2% to 65%, thereby demonstrating that it is feasible to sequence large regions of
circulating DNA by NGS.
Thus, continuous dedication to apply NGS in clinical oncology practice will enable us to
be one step closer to personalized medicine.

2. AGING RESEARCH [5]

NGS can help unravel the biological and genetic mechanisms of aging, longevity and
age-related diseases. Using NGS genes contributing to aging and age-related diseases are being
determined.
Eg: Transcription factors of the FoxO family play a conserved role in controlling longevity
downstream of the insulin pathway. FoxO orthologs are already known to extend life span in
invertebrates and SNPs in the FoxO3gene are associated with extreme longevity in humans.
Genome-wide analysis of FoxO3 binding sites in neural stem cells using next-generation
sequencing technologies identified a network of FoxO3 targets involved in stemness and
aging.
This is possible by following ways:
a. Genome sequencing and resequencing: Genome resequencing is a powerful approach to
identify genetic variants associated with longevity and/or age-related diseases. Already a
number of studies have examined the association between gene variants and human
longevity. For example, two recent studies in different populations reported gene variants in
FOXO3Aassociated with human longevity [6].
GWAS of longevity and age-related diseases using resequencing techniques will play a
pivotal role to identify new alleles that determine longevity, susceptibility to age-related
diseases and how environmental factors interact with genes to influence these phenotypes.
b. Transcriptional profiling: It has helped to identify biomarkers of aging. NGS platforms
allows researchers to characterize the aging transcriptome with exceptional resolution and
identify transcripts associated with age as well as with life-extension due to genetic or
environmental interventions in order to provide new insights about aging and its underlying
molecular and genetic mechanisms.
c. DNA-protein interactions (ChIP-Seq): Several studies have highlighted the importance of
using ChIP-Chip, by itself or in combination with other approaches, in aging research. One
of these discoveries was the finding that histones are modified at the telomeres in senescent
human cells.
d. Sequencing the epigenome: Epigenetics represents chemical alterations of the DNA and
histones that impact on function. Such alterations have been suggested to play an important
role in aging. Whole-genome analysis of epigenetic marks at the finer resolution delivered by
NGS platforms promise to be of great value to biogerontologists. Epigenetic signatures might
also be useful to identify biomarkers of aging and age-related diseases, potentially leading to
improved diagnosis and risk prediction of the latter.

Fig: Employing NGS platforms to study age-related changes. During the course of an organisms lifetime, a
number of genomic changes occur. NGS allows these changes to be quantified at a whole-genome level. Changes to
be DNA, from single nucleotide mutations to large chromosome rearrangements, can be detected (A). Likewise,
genome-wide epigenetic changes across the lifespan (or between different lifestyles or diets) can be assayed. Lastly,
transcriptional changes with age can be quantified with unprecedented accuracy using NGS (C). Mouse and human
figures were drawn using fonts by Alan Carr.

3. DRUG DISCOVERY
Utilizing NGS, early target identification can be hastened, new genetic lesions associated
with disease can be found, and overall development times associated with therapeutics and
diagnostics to newly identified targets can be significantly shortened.
Broad applications of NGS to drug discovery [7]:
1.
Mutation detection: personalized medicine
2.
ChIP-Seq: target identification and/or validation and compound profiling for epigenetics
3.
CNV: target identification, personalized medicine, for example, cancer
4.
Exome sequencing: target identification and/or drug resistance studies, biomarker
5.

discovery
RNA-Seq: target identification and/or validation by studying differential gene or miRNA

6.
7.

expression between normal and diseased tissue

HITS-CLIP: study of RNAprotein interactions
Ribosome profiling: target identification by measuring protein translation rates using

8.
9.

sequencing to identifying ribosomal footprints.

Small RNA sequencing (e.g. miRNA): biomarker discovery
Bacterial genome sequencing: target identification, validation and diagnostics to identify
new strains and mechanisms of drug resistance.

4. ANTIBODY ENGINEERING [8].

Antibody display libraries derived from human PBMCs or hybridomas immortalized from B cell
populations have been successfully used in recent decades to isolate binders against a wide range
of targets, despite a lack of detailed knowledge of the repertoires (34, 35). With the advent of
NGS, analysis of the natural nave repertoires from which libraries have been constructed has

become possible. NGS can reveal the sequence space occupied by antibodies in their recognition
of antigens.
5. ENVIRONMENTAL DNA RESEARCH [9].
The analysis of environmental DNA through the use of specific gene markers such as speciesspecific DNA barcodes has been a key application of NGS technologies in ecological and
environmental research. NGS technologies have facilitated analysis of environmentally derived
samples from a variety of ecosystems including freshwater, marine, soil, terrestrial and gut
microbiota. The majority of these studies seek to answer the question of what is present in a
given environment. Through the use of NGS platforms, researchers have been able to observe the
slight changes in community structure that may occur following anthropogenic or natural
environmental fluctuations.
Several studies have analyzed soil bacterial diversity by examining 16S rDNA amplicons.
Results suggest that agricultural management of soil may significantly influence the diversity of
bacteria and archaea. Other studies have focused on soil fungal diversity in both forest and
agricultural settings by analyzing ITS amplicons.
Marine environments have also been the subject of ecological research employing NGS
technology. Analyses of marine bacterial communities have been conducted using 18S rDNA and
16S rDNA amplicons. Frias-Lopez et al studied microbial community gene expression in ocean
surface waters through transcriptomic sequencing analysis of cDNA libraries. Mou et al.
investigated functional assemblages within seawater through a NGS analysis of functional
metabolic gene regions within bacterioplankton. Marine eukaryotic microbiota was investigated
through NGS analysis of 18S rDNA amplicons.

6. Forensic research [10].

NGS technology has become an important analytical tool for many forensic researchers. It is
being applied to simultaneously analyzing multiple loci of forensic interest in different genetic
contexts, such as autosomes, mitochondrial and sex chromosomes. Furthermore, NGS
technology has potential applications in many other aspects of research. These include DNA
database construction, ancestry and phenotypic inference, monozygotic twin studies, body fluid
and species identification, and forensic animal, plant and microbiological analyses.

Fig: Forensic analysis by next-generation sequencing

NGS will potentially influence many aspects of forensic science, including short tandem repeats (STRs) and
microRNA analysis, monozygotic twin and mixed stain recognition, Y chromosome and mitochondrial wholegenome studies, forensic microbiological analysis, multiple species identification, and ancestry and phenotype
inference. More importantly, high-throughput screening techniques have generated large amounts of data,
facilitating a systematic understanding of relationships between molecular components. Therefore, comprehensive
genome-wide analysis, in combination with the techniques of genomics, proteomics, transcriptomics and
epigenomics, will provide new insights in the field of applied forensics.

Through applying NGS technology, multiple results can be obtained simultaneously from
biological evidence samples collected from crime scenes, such as STRs, single nucleotide
polymorphisms (SNPs) of autosomes, sex chromosomes and mitochondrial genomes, as well as
epigenetic information. By integrating all the information, the evidence samples can be used not

only for suspect identification but also for inferring the criminal suspects physical,
psychological and geographical characteristics, as well as the source population.

Fig: Diverse range of information can be obtained by NGS of biological evidence samples collected from
crime scenes

7. IN FUNCTIONAL GENOMICS RESEARCH [11].

1. Genome annotation and gene expression profiling: The next generation sequencing-based
SAGE method, termed DeepSAGE, greatly simplifies the sample preparation procedure by
removing the cloning step and replacing it with emulsion PCR-based amplification; the
sequencing is conducted by the 454 protocol that allows multiple samples to be sequenced
in a single run at a high depth. Nielsen et al. applied the DeepSAGE protocol to the analysis
of the transcriptome of the potato and showed that it was efficient at detecting rare
2.

transcripts.
Small ncRNA discovery and profiling: ncRNAs are RNA molecules that are not translated
into a protein product. This class of RNAs includes transfer RNA (tRNA), ribosomal RNA
(rRNA), small nuclear and small nucleolar RNA, and microRNA and small interfering RNA
(miRNA and siRNA). Recent research has implicated microRNAs, approximately 21nucleotide-long RNA molecules, as crucial posttranscriptional regulators of gene expression
in both animals and plants. High-throughput sequencing of small RNAs provides great
potential for the identification of novel small RNAs as well as profiling of known and novel
small RNA genes.

3.

Protein coding gene annotation using transcriptome sequence data: Next-generation

sequencing technologies have the potential for providing much deeper coverage of EST
libraries. A recent study used laser capture microdissection to isolate transcripts from the
shoot apical meristem of Z. mays followed by cDNA library construction and 454
sequencing of ESTs. The study used a cis-alignment method to annotate more than 25,000
genomic sequences from maize and detect transcription from 400 orphan genes, most of

4.

which had not been detected using other approaches.

Detection of aberrant transcription: Large-scale transcriptome sequencing studies provide a
novel means for detecting genome rearrangements in the transcribed portion of the genome.
An elegant gene identification signature analysis using paired-end ditag transcriptome
sequencing methodology has been developed for the detection of gene fusions and other
aberrant transcripts in cancers. The approach involves generation of 18-nt-long tags from
both ends of a transcript, which are then concatenated and sequenced by the 454 technology.
This strategy is particularly useful for detecting fusion events in cancers, as well as actively

5.

transcribed Pseudogenes that are readily distinguishable from their source genomic loci.
Analysis of epigenetic modifications of histones and DNA: The NGS technologies offer the
potential to accelerate epigenomic research substantially. To date, these technologies have
been applied in several epigenomic areas, including the characterization of DNA
methylation patterns, posttranslational modifications of histones, and nucleosome

6.

positioning on a genome-wide scale.

Study of DNA accessibility and chromatin structure: NGS technologies have been applied
to mapping out the positions of nucleosomes and other determinants of DNA accessibility

8. AGRICULTURAL RESEARCH [12].

NGS technologies have several potential applications in crop genetics and breeding, including
the generation of genomic resources, marker development and QTL mapping, wide crosses and
alien gene introgression, expression analysis, association genetics and population biology, as
shown here. For instance, sequencing of genomic DNA including bacterial artificial
chromosomes (BACs), reduced representation of genome (RRG) or cDNA from the reference
genotypes using NGS technologies can provide genomic resources such as ESTs, gene space and
genome assembly.
These resources have a direct impact on understanding the genome architecture for crop genetics.
Another application of NGS is in parental genotyping of mapping populations or of wild
relatives, which can accelerate the development of molecular markers, e.g. simple sequence
repeat (SSR) and single nucleotide polymorphism (SNP) markers. These markers can be used to
construct genetic maps, to identify QTLs and to monitor alien genome introgression in the case

of wide crosses. These QTL-associated markers for a trait of interest can then be used in
selecting progenies carrying favorable alleles via marker-assisted selection (MAS). To develop
the functional or perfect gene-based marker, NGS of cDNAs of contrasting genotypes for the
trait of interest can be used to identify candidate genes involved in or associated with the trait.
The expression mapping of these candidate genes, together with phenotyping of the segregating
populations developed from the contrasting genotypes, will provide expression QTLs (eQTLs)
and markers associated with these eQTLs should thus serve as the perfect markers for MAS in
crop breeding. Another important application of NGS is in association genetics or population
biology, where either genomes or pools of PCR products of thousands of candidate genes can be
sequenced in hundreds of individuals using barcodes. The sequence data obtained could then be
used to identify SNPs or haplotypes across genes or genomes for use in association genetics
and/or population biology.

Fig: Overview of NGS applications in crop genetics and breeding

Table: Applications of NGS technologies in plant genetics and breeding

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