Next Generation Sequencing Technology: Revolutionizing Diagnostics & Molecular Therapies

Next Generation Sequencing Technology Revolutionizing Diagnostics & Molecular Therapies

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Jan 24

Genomics, proteomics, and gene regulation are complex fields of human biology that hold answers to evolution, disease propagation and manifestation. The discoveries of DNAs and RNAs opened up horizons not only in identifying genetic cause of diseases or inventing new technologies and therapies for disease management, but also in looking for life in the outer space.1

The continuously evolving genetic therapies are the result of collaborative efforts from specialized fields of genome sequencing, gene editing, nanomedicine, bioinformatics, microbiometrics, and many other disciplines, depending on the research. A universe of knowledge is embedded in the coded letters of DNA and the greatest tool to untap this repository since the last century has been the Next Generation Sequencing (NGS) technology.

The first-generation DNA sequencing, or Sanger sequencing, named after its inventor, Fredrick Sanger, was invented in 1977, and it played a crucial role in completing the Human Genome Project in 2003.2 It took 13 years and $3 billion to generate the first sequence of the human genome.3 Now, an individual can obtain her full genome sequence within a day, and the cost is only $600, which used to be $1000 in 2014 and an estimated $1 million in 2007!4,5

This has been possible with rapid inventions in NGS, which is a parallel sequencing technology that has high-throughput, is scalable, and is extremely fast. The findings from NGS have evolved the queries by the scientists and revolutionized the study of biological sciences to an unfathomable level where the magnitude of disease-causing elements encompasses genetic, environmental, and behavioral factors. The significance of NGS tests, the analysis and interpretation of the results, and the subsequent application of targeted molecular therapies in the form of gene regulation are beyond the scope of regular medical practitioners. It requires the expertise of scientists from across many disciplines, like molecular medicine, genetic engineering, data analysis, bioinformatics, etc., with the parallel application of machine learning, particularly deep learning methods, for comprehension of the enormous data generated from the tests.6

Genome information is complex, and the data gathered from sequencing an individual’s full genome, large section of genome, and targeted region of DNA or RNA to detect disease-causing genes7 as well as discovering genes assigned to specific cellular functions are, at the heart of biological and medical research for novel therapeutics and proposed preventative measures. Considering the previous Sanger sequencing could only determine smaller and single fragments of DNA with a maximum of 900 bases, NGS being high-throughput is capable of parallelly sequencing multiple fragments of DNA and generating hundreds of millions of readable sites in the DNA in less time and for less cost.8,9

NGS can also detect associations between specific diseases and gene mutations or gene regulation that is dependent on other factors. However, cost is a consideration, and so is the implication of testing.

What is Epigenetics and its relationship with Human Genetics?

The same disease is not homogeneously manifested among individuals.8 Epigenetics involves the genetic control of turning genes “on” or “off” by factors other than the DNA itself. It regulates how the DNA sequence is ‘read’, is reversible, and does not cause a change in the DNA sequence.

A change in genetic sequence can alter which protein will be made, but epigenetic modification alters access to specific sites in the DNA and controls gene expression by switching genes “on” and “off.” Instead of instructing which protein is to be made, epigenetics control how often and when proteins are to be synthesized. This is broadly done through DNA methylation, histone modification and non-coding RNAs. DNA methylation turns genes “off” by placing a chemical group on specific sites of a gene that blocks the protein that was supposed to attach to that particular site to ‘read’ the gene for gene expression. Typically, genes can be turned “on” by demethylation, where the chemical group can be removed.10

Histones are complex proteins that have DNA strands wrapped around them to form bead-like structures. When these bead-like structures are tightly packed in such a way that the proteins that ‘read’ the gene do not get access to that gene, it structurally turns the gene “off”. Chemical groups can be added or removed to make histones loosely or tightly packed to switch genes “on” or “off.”10 Non-coding RNA has the ability to attach itself to coding RNA and break down the coding RNA to stop it from transcribing proteins. DNA is used for making both coding and non-coding RNAs.

So, DNA methylation, tightly packed histones and non-coding RNA are the epigenetic factors that turn genes “off” and thus control gene regulation.10

Although all our cells have the same gene, our body has 200 types of cells. This is attributed to epigenetics, which is involved in normal cellular processes and differentiates the cells into heart cells, nerve cells, muscle cells, etc. from the time of conception. Age affects epigenetic regulation, with newborns having the highest level of DNA methylation to ensure specific cells are developed by activating certain genes and deactivating other gene expressions. Behaviors like smoking affect epigenetics, where parts of specific genes have less DNA methylation, leading to higher gene expression, which can be reversed by quitting smoking.10 Infections sometimes switch genes “off” that weaken the immune system. Cancers overall usually lessen the levels of DNA methylation, but in some cases, they increase, which can inhibit the protective tumor suppressor genes11 leading to different kinds of cancer. The pregnancy period also influences genetic expression in later life.10

NGS, specifically the third-generation NGS technologies, have the ability to detect epigenetic changes in DNA methylation and histone modifications.

What are the types of Next Generation Sequencing Technologies?

Currently, there are second-generation and third generation NGS technologies, with the second-generation being used the most as it is the cheapest and fastest. Despite that, the development of third-generation NGS technologies continues to grow, especially since prices are dropping.12 NGS generates a large volume of data and there is already data available on open-source platforms with which new data can be cross-matched.

The most fundamental basis of NGS is first knowing what the researchers are looking for. The indications of application of NGS sequencing include pure research, drug development, biomarker analysis, therapeutic decision-making, studying the microbiome, identifying novel pathogens and rare diseases.12,13

A lot of focus was given to second-generation NGS technologies, which now have high accuracy, are low-cost, can sequence short fragments (200–300 bases long) parallelly very fast, focus on the exome and not the full genome, and are best in clinical settings with improved data analysis. However, they are unsuitable for reading large sections of DNA.

Third-generation technologies (Fig-1) are being developed for research purposes to read larger sections of DNA or the whole genome with little reference data. This is also used for studying the gene-regulating epigenetic markers of DNA methylation and histone modification, transcriptomics- which involves everything about the RNA, and metagenomics- which is the study of the full genome sequence of all organisms in a bulk sample.12,14

Summary of Third-generation Sequencing

However, the accuracy of third-generation NGS technologies is still challenged, and the signals from individual fragments are weak.12

What are some of the well-known NGS Technologies available in the market?

The big names in second-generation NGS technology are Illumina (Solexa) sequencing, Illumina HiSeq X Ten Sequencer, Illumina NovaSeqX series, Roche 454 sequencing, Ion torrent: proton/PGM sequencing, solid sequencing, GenapSys sequencing Platform, and BGI Groups DNBSEQ-T7.12,16

Among the third-generation NGS techniques, Pacific Biosciences’ PacBio Revio system, Oxford Nanopore Technologies’ MinION, and Oxford Nanopore sequencing chemistry are well known.12

The NGS-based methods available for epigenetic analysis are:

Methyl-seq uses single-nucleotide resolution to identify the methylation status of the genome.

ChIP-seq is commonly used to map histone modifications and transcription factors.

ATAC-seq determines regions of chromatin accessibility and maps DNA-binding proteins to identify active promoters and enhancers.17

Universal Nicking Enzyme-assisted Sequencing (UniNicE-seq) and Nicking Enzyme-assisted Viewing and Sequencing (NiCE-view-seq) captures and reveals open chromatin sites and transcription factor occupancy at single nucleotide resolution.18

The market leader in NGS technological tools is Illumina and it controls 80% of the global DNA sequencing market.5 Thermo Fisher Scientific, Perkin Elmer, Agilent Technologies in the US, and BGI Group in China are prominent players.19

What are the challenges of NGS and how is it interpreted?

All NGS technologies generate millions of data points because NGS’ are basically millions of tests within one. There are also challenges in determining the accuracy of the data. The cornerstone of managing the data is improved mathematical and statistical models to accommodate the growing data.8

Since NGS generates enormous amounts of information, it is not feasible to impose existing regulatory structure on it that is available for conventional diagnostics where one investigation is standardized for one specific disease. Understanding the complexity, the Food and Drug Administration in the US involved all the stakeholders relating to the NGS industry to come to a consensus and create a regulatory framework. This framework is kept flexible for the ever-changing and ever-expanding technology so that newer NGS techniques can leverage the updated data, crowd-sourced data, and open-source software to support their development.7

The interpretation of NGS is continuously evolving. Databases available online provide list of clinically significant genes. Upon finding a new variant, it is mandatory to review the updated literature available to evaluate whether the emerging variant can be flagged as a variant of concern. 20

NGS is a major focus area of research as it detects new disease-related genes almost every other month. It is now at the core of medical research to develop therapies and preventative approaches. This relatively new domain is identifying multiple disease factors at genomic, epigenomic, and transcriptomic levels.18

There is a long list of genetic diseases already discovered and new rare ones are being discovered frequently. Other than developing therapies and diagnosing them early, NGS- based genome sequencing among large groups of people as a routine- based screening tool will help determine common genetic traits and combat progressive common disorders based on gene mutation. IndiGen in India is carrying out such a project to build their database.24

Genetic diseases discovered recently from NGS technology

What is the NGS Technology scenario in Bangladesh?

Human genome sequencing in Bangladesh is not done as a regular test or in regular diagnostics (Fig3). During the time of Covid- Institute of Epidemiology, Disease Control, and Research (IEDCR), the International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b), the Child Health Research Foundation (CHRF), and the Institute for Developing Science and Health Initiatives (ideSHi) built a consortium to work closely to monitor genomic variants of the SARS-CoV-2 virus. IEDCR-ideSHi used the nanopore MinION and MiSeq Illumina platforms; ICDDR-B used the MiSeq Illumina platform; and CHRF utilized the NextSeq Illumina platform for the virus’ genome sequencing.25,26 CHRF recently added Sanger sequencer SeqStudie Flex to ndetect bacterial species infecting children.

Organizations like icddr,b, Bangladesh Agricultural Research Institute, Bangabandhu Sheikh Mujib Medical University, North South University, and Globe Biotech have Next Generation Sequencing (NGS) facilities, but their services are not commercially available. Jashore University of Science and Technology has built a laboratory with QuantStudio 3 Real-Time PCR Systems for Real-Time PCR, an Ion GeneStudio S5 Semiconductor Sequencer for Next Generation Sequencing (NGS), a SeqStudio Genetic Analyzer for 16S rRNA sequencing, and plans to do whole genome sequencing and 16S ribosomal RNA (rRNA) sequencing in the future.27

Bangladesh Scenario in Funding for NGS

As of March 2023, Bangladesh has 22 NGS machines out of which 10 are from ONT, eight from Illumina, and four from Thermo Fisher. 100% spending on NGS capacity comes from private, and external partner-based funding (Fig-2). The external partners include- Wellcome Trust, UK; Bill and Melinda Gates Foundation; USAID; Foreign, Commonwealth & Development Office (FCDO); Centers for Disease Control and Prevention (CDC) in Atlanta; the Wellcome Sanger Institute (UK); and Washington University in St. Louis. Although, human genome sequencing is lagging behind, significant impact has been observed in agriculture and food security.28

The private sector conducts 40% of all NGS, the public sector 30%, and academic institutions the remaining 30%. Genome studies of all priority pathogens (influenza and TB) are now being done in the country.

The major challenges in NGS application in Bangladesh are the availability and cost of laboratory supplies and consumables (reagents, PPE, etc.), labor costs, and staff training. Even if the private and external sectors invest in developing the infrastructure, a preliminary focus must be given to training skilled people in data processing, quality assurance, bioinformatics to store the data, and data analysis.29

The NGS market size was $13 billion globally in 2022 which is estimated to grow to $27 billion by 2027 with a growth rate of 15.7%. The main reason for the growth is the continuous decline in sequencing cost and high incidence of cancers. Main cost of NGS is investments in equipment, platform, consumables, services, and bioinformatics.19

The snapshot of NGS Technology capacity, output, utilization, and quality assurance in Bangladesh

The expansion of NGS technology in Bangladesh is at a nascent stage. NGS application is key to molecular diagnostics and drug discovery, especially precision medicine.19 Even though NGS diagnostics are getting cheaper in the West, they are not perceived to be cheap in Bangladesh. For better prognosis and with the promise of early recovery if diseases are detected early with NGS, the eventual healthcare cost burden will lessen. We must focus on bringing this technology to the forefront in our country, and since we have a large population, investors will recover their costs within a very short period of time thanks to economies of scale.

Author of this article:
  • Dr. Maliha Mannan Ahmed MBBS (BMC), MBA (ULAB) & Masters in Healthcare Leadership (Brown University), Executive Editor of The Coronal
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