ASSIGNMENT BY JIMOH ADEDAYO DANIEL

BCH708

BIOTECHNOLOGY

APPLICATION OF NEXT GENERATION SEQUENCING IN ONE HEALTH APPROACH

Next-Generation Sequencing (NGS), also known as high-throughput sequencing, is a technology

that enables the rapid and parallel sequencing of millions to billions of DNA or RNA fragments

simultaneously. Unlike traditional Sanger sequencing, which processes one DNA strand at a time,

NGS increases speed, scalability, and throughput, allowing for the sequencing of entire genomes

in days. This method has transformed fields like genomics, personalized medicine, and

bioinformatics by allowing researchers to study genetic variations, mutations, gene expression,

and more. NGS is considered the gold-standard sequencing technology, allowing for the

interrogation of hundreds to thousands of genes at once in multiple samples and the discovery

and analysis of different genomic features in a single sequencing run. It also offers lower sample

input requirements, higher accuracy, and the ability to detect variants at lower allele frequencies

than traditional methods.

TYPE OF NEXT GENERATION BASE OMICS INCLUDES

[Link] SEQUENCING This is a process that involve the analysis of DNA using Three

approach WHOLE GENOME SEQUENCING Which involve sequencing and determination of

complete DNA sequencing of individual genome This whole genome sequencing has two type of
sequencing approach base on genome size, Large whole genome sequencing deciphering large

genomes of > 5MB usually eukayotics and small whole genome sequencing deciphering <5MB

usually prokaryotic. Whole-Genome Sequencing (WGS): Sequences the entire genome, including

~98% non-coding DNA. Ideal for discovering novel variants and structural changes. Whole-Exome

Sequencing (WES): Targets only the exome (protein-coding regions, about 1-2% of the genome)

where most disease-causing mutations occur. More cost-effective than WGS for clinical use.

Targeted Sequencing: Focuses on specific genes or regions of interest, such as cancer-related

panels. RNA Sequencing (RNA-Seq): Sequences RNA to study gene expression, alternative

splicing, and transcriptomes, rather than the static DNA genome.

[Link]

Transcriptomics is the study of the transcriptome, which refers to the complete set of RNA

molecules (transcripts) produced by the genome of an organism, cell, or tissue at a specific time

or under particular conditions

Types of Transcriptomics Methods

Transcriptomics encompasses various approaches based on scale and technology:

 Bulk Transcriptomics: Analyzes RNA from a population of cells, averaging signals.

Common methods: RNA-Seq and microarrays

  .Single-Cell Transcriptomics (scRNA-Seq): Profiles individual cells to uncover

heterogeneity, using droplet-based (e.g., 10x Genomics) or plate-based methods.

 Spatial Transcriptomics: Combines gene expression with spatial location in tissues, via

techniques like Visium or MERFISH.

 Other Specialized Types: Includes non-coding RNA-seq, isoform sequencing (for

alternative splicing), and time-course transcriptomics for dynamic studies.

3. EPIGENOMICS

Epigenomics is the comprehensive study of epigenetic changes across the entire genome in a cell

or organism, focusing on modifications that influence gene expression without altering the

underlying DNA sequence.

Types of Epigenomics Methods

Epigenomic methods vary by target and scale, broadly categorized as:

 DNA Methylation Profiling: Includes whole-genome bisulfite sequencing (WGBS) for base-

resolution mapping, reduced representation bisulfite sequencing (RRBS) for CpG-rich

regions, and affinity-based methods like MeDIP-Seq.

 Histone Modification and Protein-DNA Interaction: ChIP-Seq and variants (e.g., ChIP-exo)

use antibodies to pull down modified histones or transcription factors.

  Chromatin Accessibility and Structure: ATAC-Seq (using transposase to tag open

chromatin) and Hi-C (for 3D interactions via proximity ligation).

 Single-Cell and Spatial Epigenomics: scATAC-Seq or scBS-Seq for cell-level resolution;

spatial methods like MERFISH for tissue context.

 Multi-Omics Integration: Combines epigenomics with genomics/transcriptomics, e.g.,

NOME-Seq for methylation and accessibility.

4. Metagenomic sequencing is the process of sequencing all the DNA present in an environmental

sample to study the community of microorganisms within it.

THE APPLICATION NEXT GENERATION SEQUENCING IN ONE HEALTH APPROACH

[Link] MONITORING AND SURVEILLANCE.

One of the most critical applications of NEXT GENERATION SEQUENCING and bioinformatics

within the One Health framework is disease monitoring and surveillance. Next-generation

sequencing (NGS) technologies enable the rapid and comprehensive sequencing of pathogen

genomes from diverse sources, including human, animal, and environmental samples. This

capability facilitates the identification and tracking of pathogens with high precision, especially

using NGS techniques, which provide crucial information on specific strains and their genetic

characteristics. This is particularly important for tracking the spread of zoonotic diseases, where

pathogens jump from animals to humans. By comparing genomic sequences from different hosts

and locations, researchers can trace the transmission pathways of infectious agents, understand

their evolution, and identify potential reservoirs of infection, as highlighted by the World Health

Organization

Moreover NGS enables the early detection of outbreaks by allowing for the identification of

pathogens at very low levels, often before clinical symptoms appear in affected populations. This

early detection is crucial for implementing timely interventions to prevent outbreaks from

becoming widespread. Bioinformatics tools further facilitate the analysis and interpretation of

large-scale genomic data, helping to rapidly identify emerging threats.

Next-generation sequencing and bioinformatics are crucial in disease prevention by revealing

genetic factors influencing susceptibility and resistance. This knowledge can be used for risk

prediction and personalized medicine, identifying genetic markers associated with increased

susceptibility to diseases in humans and animals. This allows for the development of personalized

prevention and treatment strategies, such as vaccination or health monitoring. Genomics also

aids in designing effective vaccines by providing detailed information on pathogen genomes,

enabling targeted vaccine development. Bioinformatics tools predict vaccine efficacy and identify

optimal antigen candidates.

Next-generation sequencing and bioinformatics are crucial for health promotion by enhancing

our understanding of the interactions between genomes, environments, and lifestyles. This

knowledge is essential for developing interventions that promote health and well-being across

populations. Research into genotype-environment interactions helps understand how genetic

factors influence responses to environmental exposures, such as pollutants, diet, and climate

change. By integrating genomic data with environmental data, bioinformatics analyses can

identify populations at risk of adverse health outcomes due to specific environmental factors,

guiding public health policies and interventions. Nutrigenomics studies the relationship between

an individual's genetic makeup and their response to diet, identifying genetic variations that

affect nutrient metabolism and dietary requirements. Large-scale genomic studies across diverse

populations can reveal population-specific health risks and inform targeted health promotion

strategies.
[Link] OF NOVEL CANCER MUTATIONS USING NGS

NGS technologies have been used to detect novel and rare somatic mutations in various 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, identifying a novel PML-

RARA genetic recombination undetectable with standard cytogenetic techniques. In a study by

Gui et al., they sequenced the exomes of 9 transitional cell carcinoma tumors and screened in

tumor samples from 88 individuals with transitional cell carcinoma at different stages and grades.

They also sequenced the whole exomes of 10 clear cell renal cell carcinomas and screened

thousands of genes in an additional 88 samples, discovering 12 new mutated genes.

Hereditary cancers account for 5%-10% of all cancers, and genetic testing has been used for over

ten years in the US and Europe. Sanger-based sequencing is the most widely used method for

detecting mutations, but it is time-consuming, high-cost, and low-throughput due to the large

size of genes. The development of next-generation sequencing (NGS) has provided numerous

opportunities for genetic testing, as demonstrated by Walsh et al.'s study using target region

capture and NGS to detect 21 genes associated with hereditary breast and ovarian cancer.

Commercial testing for BRCA1 and BRCA2 in the US and Europe uses polymerase chain reaction

(PCR) amplification and Sanger sequencing, with multiplex ligation-dependent probe

amplification (MLPA) added for large exonic deletions and duplications.

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, found previously undetected

mutations in 52 probands. And the reduced sequencing costs and turnaround time made the

approach even more practical in clinics, introduced a method that used long-range PCR plus NGS

to detect BRCA1 and BRCA2 and demonstrated that it was useful for BRCA testing. For a small

sample size, the method is combined with the Miseq or Ion torrent platform. Moreover, this

approach may be more flexible and economic than a capture strategy. The use of NGS in genetic

testing for hereditary cancer syndromes will be the first and closest step for its transition into

clinical practice. It is more exciting that whole-genome or whole-exome sequencing of malignant

tumors has been used in several clinical trials for personalized therapy.

NGS for personalized cancer treatment

Apart from identifying genetic and novel somatic mutations, another use of NGS is to improve

rationally designed individualized medicine. To date, many studies have applied NGS for

personalized treatment of cancer. 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 non-small 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
patient's therapeutic schedule. By using Affymetrix SNP arrays, Illumina mRNA expression arrays

and sequencing method, Holbrook observed the KRAS, ERBB2, EGFR, NET, PIK3CA, FGFR2,

and AURKA gene mutations in gastric cancer, and their data suggested targeted therapies for

gastric carcinoma that would benefit 11 of the 50 patients in their study. Roychowdhury scientist

reported the first study of whole-genome and whole-exome plus transcriptome sequencing in

personalized treatment. In the study, they tested the integrated strategy in several patients, and

the resultant sequencing data were discussed by the Sequencing Tumor Board, a panel of clinical

and genome sequencing experts, to determine the appropriate treatment. Next they used the

strategy in actual patients and sequenced tumor and normal DNA. Two patients, one with

colorectal cancer and the other with melanoma, who were previously treated unsuccessfully,

were enrolled into the pilot study. The integrative sequencing and analysis were completed

within 4 weeks after biopsy. In the patient with colorectal cancer, variations

in CDK8 and NRAS were found and considered the potential targets to match to a clinical trial in

the future. For the patient with melanoma, a structural rearrangement in CDKN2C and HRAS was

detected, prompting the Sequencing Tumor Board to suggest combined treatment with PI3K and

MEK inhibitors for this patient

3. WHOLE GENOME SEQUENCING (WGS) FOR EPIDEMIOLOGY & ANTIMICROBIA RESISTANCE

WGS to develop novel antibiotics

WGS has become a crucial tool in drug development, particularly in the context of tuberculosis

(TB), a global public health emergency. In 2005, the first published use of 454 pyrosequencing

was to identify the F0 subunit of the ATP synthase as the target of bedaquiline, which became

the first representative of a novel class of anti-TB agents to be approved in 40 years. This enabled

researchers to sequence this gene in phylogenetically diverse reference collections to ensure its

conservation across Mycobacterium canettii and various lineages and species that comprise the

Mycobacterium tuberculosis complex (MTBC), the causative agents of TB.

The early elucidation of resistance mechanisms using WGS has implications for the design of

clinical trials. If resistance mechanisms only result in marginally increased minimal inhibitory

concentrations (MICs) compared with the wild type MIC distributions, more frequent dosing or

higher doses could be employed in clinical trials to overcome this level of resistance.

WGS has become increasingly important during clinical trials, particularly in distinguishing

exogenous reinfection from relapse of the primary infection, which is crucial in assessing the

efficacy of the drug or regimens under investigation. Traditional epidemiological tools do not

always provide the necessary resolution for this purpose due to their limited interrogation of the

complete genetic repertoire of an organism. WGS will become the gold standard for clinical trials

of new anti-TB agents and other infectious diseases associated with recurrent disease
WGS TO STUDY THE EMERGENCE OF ANTIBIOTIC RESISTANCE

WGS has been used to study the evolution of antibiotic resistance in real-time under various

conditions, such as maintaining constant antibiotic pressure in Escherichia coli and studying

ethambutol resistance in MTBC. Toprak et al. developed a microbial cultivation system to

monitor resistance evolution, while Safi et al. used antibiotic-containing solid medium to study

ethambutol resistance in MTBC. The latter study demonstrated that ethambutol resistance

emerges in a stepwise process and highlighted a novel resistance mechanism, such as a

synonymous mutation in Rv3792 increasing ethambutol MICs by increasing the expression of the

downstreamgene

WGS has also played a role in measuring the rate at which resistance emerges. Ford et al. found

that lineage 2 MTBC isolates acquire rifampicin resistance at an elevated rate compared to

lineage 4 strains, suggesting that patients infected with drug-susceptible lineage 2 isolates have

a 22-fold increased risk of developing multi-drug resistance (MDR) by the time they are initially

diagnosed.

WGS has also clarified factors that have allowed the spread of drug-resistant MTBC, such as

mutations in the α and β’ subunits of the RNA polymerase that alleviate the fitness costs of

resistance to rifampicin. These compensatory mutations are widespread in MDR MTBC isolates

in high-incidence countries and are associated with ongoing transmission. Therefore, the

importance of developing and deploying rapid point-of-care drug-susceptibility testing (DST)

assays to identify MDR strains is underscored.

4. NEXT GENERATION SEQUENCING FOOD SAFETY AND FOODBORNE VIRUS SURVEILLANCE

Next-generation sequencing (NGS) is revolutionizing food safety by providing sensitive detection

and high-throughput capabilities. Laboratory networks like GenomeTrakr Network and PulseNet

use whole-genome sequencing to identify foodborne pathogens like Salmonella, Listeria, E. coli,

Campylobacter, Vibrio, and Cronobacter. These networks collect and share genomic and

geographic data, enabling public health responses. PulseNet coordinates labs worldwide to

identify foodborne illness outbreaks with known pathogens, alerting the public and identifying

gaps in food safety systems. Early detection of outbreaks can limit the spread of contaminants

from microorganisms.

CURRENT APPROACHES IN FOOD VIROLOGY

The first viral food-borne outbreaks in the late nineteenth century were attributed to the lack of

routine laboratory testing for noroviruses and HAV, which was limited to electron microcopy.

This led to studies exploring the use of indicator organisms, particularly bacteriophages, as

surrogates, with limited success. Since the sequencing of the complete genome of the prototype

norovirus in 1990, molecular biology methods have become the mainstay in food virology,

providing first-time tools applicable in this field.

Next-Generation Sequencing (NGS) or high-throughput deep sequencing refer to successive

technologies developed since 2005 that allow for massive, parallel sequencing of DNA fragments.

Several studies have applied NGS for the detection and characterization of viral diversity in food
using varying strategies. Some target specific viruses or viral families, while others provide a

description of the whole RNA or DNA virome.

The first strategy is similar to the metabarcoding approach with deep sequencing of PCR

amplicons, which is limited to closely related viruses or viral families due to the wide genetic

diversity of viruses. This approach was applied to analyze the diversity of norovirus GI and GII

genotypes in naturally contaminated oysters from Japan, using PCR primers targeting the N-

terminal region of the VP1 protein commonly used to determine the norovirus genotype.

A second strategy is full-genome sequencing, using deep sequencing of PCR amplicons spanning

the entire viral genome. This was applied in clinical samples using primers that target the

conserved regions of the 5′- and 3′-end of genomic and subgenomic norovirus RNA. In food, this

strategy was applied on frozen berries linked to HAV-outbreaks in Italy, allowing the sequencing

of a nearly-complete HAV IB genome and portions of the norovirus genome closely related to the

virus identified in patients.

This study highlights the potential of NGS to help identify the etiological agents and their possible

origin in foodborne outbreaks, but also the limits posed by the detection level of the technique.

In naturally-contaminated foods, such as lettuce or meat, few reads assigned to a diversity of

mammalian viruses remain a challenge. However, due to the high amounts of sequences from

non-mammalian viruses, this approach could also help in the surveillance of circulating antibiotic-

resistance genes in phages and viruses affecting farmed species.

5. NEXT GENERATION SEQUENCING FIELD DEPLOYMENT & LMIC CAPACITY BUILDING

A bioinformatics capacity-building initiative in six East African countries focuses on three pillars:

infrastructure provision, workforce development, and institutional support. The initiative aims to

enhance the use of portable NGS technology in one health, enabling real-time, on-site pathogen

detection, empowering local health systems, and enabling democratic access to sequencing

technology. Advantages of portable NGS include reduced diagnostic lag, immediate public health

or agricultural responses, and democratic access to sequencing technology. However,

operational challenges include cost barriers, supply chain issues, technical expertise, and

sustainability concerns. Cost barriers include expensive consumables like flow cells and cold

storage, supply chain issues in many LMICs, and the need for advanced molecular biology and

bioinformatics skills. Technical expertise is also a challenge, as running NGS workflows requires

advanced skills. Without policy integration, ongoing funding, and institutional ownership, gains

in capacity can erode over time.