High-Throughput Genomic Sequencing With Reduced Cost

The diverse next-generation high-throughput sequencing technologies have significantly enhanced the speed and ease of DNA sequencing and drastically reduced the cost, bringing in the genomic revolution.

DNA sequencers play a vital role in the biotechnology industry. The ability to know the four-letter sequence of any gene or DNA segment is essential to conducting research and analyzing the genetics of individuals, organisms, plants, and animals. In the last 10 years, cost of DNA sequencing has reduced quite significantly. With every passing month, more and more people from different parts of the world are getting their DNA sequenced. Thus, not only medical practitioners but even common people, including patients now know the importance of knowing the sequence of their DNA. Early attempts to sequence DNA were cumbersome. The 1970s technologies of Sanger’s chain termination, and Maxim and Gilbert’s chain degradation were improved upon, by advances in fluorescence-based fully automated sequencing machines in 1987. An important aspect of these automated sequencers is that they use fluorescent labelling as opposed to radioactive labelling, thus completely eliminating radioactivity hazards and manual base determination. Later, many modifications were introduced in terms of instrumentation, sequencing chemistry, and sequencing enzymes. Automated DNA sequencing machines which are capable of performing sequencing with large capacities have been developed and marketed by many companies.

Sequencing has gotten faster and cheaper; and next-generation sequencing has taken it to the level of complete genome sequencing in a matter of hours. The development costs and time associated with creating state-of-the-art instruments can approach the lower range of costs typically associated with therapeutics. Automated sequencing machines and the resulting databases have led to the rapid development of computer software to assemble, manipulate, and analyze genetic sequence. This enables the exploration and characterization of the various genomes and comparisons among them. Moreover, latest technologies available in the market are capable of producing several gigaBASE of data in a single sequencing run facilitating the analysis of large chromosomes and genomes. These diverse next-generation sequencing technologies have significantly enhanced the speed and ease of DNA sequencing and drastically reduced the cost bringing in the so called genomic revolution. Inspired by the success of next-generation or second-generation sequencers, third-generation DNA sequencers based on single molecule sequencing (SMS), real-time sequencing, and nanopore sequencing have also been launched. It is hoped that these third-generation DNA sequencers, especially the nanopore-based ones will produce incredibly long read DNA sequence data far cheaper and much faster than currently possible by the second-generation sequencers.

With the exponential improvements in sequencing DNA, there has been an explosion of genomic information which is giving a greater understanding of disease. This can increase the understanding of genetic factors influencing the transmission, evolution, and pathogenesis of infectious agents. The range and scope of DNA sequencing applications has also expanded over the past few decades, shaped in part by the evolving constraints of sequencing technologies. Understanding and interpreting the human genome is a cornerstone of modern medicine, offering a wealth of information about a person’s inherited genetics risks, the antibodies they have, or how their diseases – such as cancer – have developed. The ability to sequence human genomes has vastly outpaced the ability to interpret genetic variation, which partly explains why clinical medicine has yet to wholeheartedly embrace WGS. Nonetheless, there are some areas in which DNA sequencing is already proving clinically useful. The most unexpected area of the clinical impact of DNA sequencing has been non-invasive prenatal testing (NIPT). Screening tests that were based on this strategy are adopted faster than any molecular test in history, and several million pregnant women around the world have already benefited from low-pass WGS for NIPT. An early application of WES was to rapidly discover new genes for, and to diagnose patients affected by Mendelian disorders and neurodevelopmental disorders.

Early detection of cancer is top of mind for many researchers and companies. They know that detecting cancer early can improve a patient’s chances of survival – perhaps as much as 5–10 times compared to that of a late-stage diagnosis. In practice, however, it is extremely challenging to determine if a person has a medically relevant tumor until it is advanced to a larger and more threatening stage. Thanks to advances in DNA sequencing technology, there is a new and powerful tool that may be able to identify patients with early stage cancer and help direct therapeutic strategies. Indeed, as efforts to sequence larger genomes took shape, it became clear that the scale and efficiency of each step needed to be vastly increased. Noteworthy improvements included switch from dye-labelled primers to dye-labelled terminators, technologies for linear amplification which greatly reduced template requirements and facilitated miniaturization, magnetic bead-based DNA purification techniques that simplified automation of presequencing steps, capillary electrophoresis technology which eliminated the pouring and loading of gels, while also simplifying the extraction and interpretation of the fluorescent signal, and adoption of industrial processes to maximize efficiencies and minimize errors (e.g., automation, quality control, standard operating procedures, and so on).

Indian market
The Indian market in 2015 for DNA sequencers is estimated at Rs. 238.8 crore. It may be segmented as capillary sequencers and NGS. The capillary sequencers market is divided in the ratio of 53 percent instruments and 47 percent consumables, whereas the NGS constitutes 44 percent of instruments and 56 percent consumables. This is very different from 2016, when the market for NGS was skewed as 70 percent instruments and 30 percent consumables. Last year, in October 2016, MedGenome, a Bengaluru-based company into genomics research and diagnostics procured the Illumina Hiseq X Ten machine, a population scale sequencer for USD 5 million approximately. 2017 onwards the ratio is gradually coming back to a 50:50 for instruments and consumables. Other companies who procured NGS instruments in 2017 include Institute of Genomics & Integrative Biology (IGIB), ISBT, National Institute of Biomedical Genomics (NIBMG), and Strand Life Sciences.

Moving forward, this market is expected to do well, as buyers as MedGenome penetrate tier II and tier III cities utilizing the capital they have raised to expand the clinical genomic testing market. While having raised funds for USD 30 million in financing led by Sequoia India and Sofina s.a. in August 2017 and USD 10 million from HDFC Ltd., HDFC Life and HDFC Asset Management in March 2018, the company has announced its plans to further democratise the critical genetic tests like noninvasive prenatal screening (NIPT) and new-born genetic testing.  The company also plans to establish more genetic centers in hospitals across the country to support clinicians and to enable patients to make informed decisions.

A massive genome-sequencing effort is coming to India
A UK-based genomics data platform and an American genetics company have recently (March 2018) collaborated to create the world’s largest project of its kind to study the Indian population. Cambridge-headquartered Global Gene Corp’s (GGC) new multi-year tie-up with Regeneron Genetics Center (RGC), a wholly-owned subsidiary of New York-based Regeneron Pharmaceuticals Inc, is aimed at finding innovative diagnosis and therapies for rare diseases. According to Deepak Bagla, managing director and CEO of Invest India, the country’s investment promotion and facilitation agency which is supporting GGC to build world-class capabilities in Mumbai and Ahmedabad believes that genomics will help India achieve a paradigm shift in healthcare, and this latest collaboration marks a step forward in the Indian government’s Healthcare for All plans, particularly with Ayushmann Bharat initiative.

Genetic evidence has revolutionized scientific discovery and drug development in recent years by providing clear links between certain genes and disease. This genomics revolution is crucial to the delivery of improved healthcare for all. A deeper understanding of the genetic architecture and disease burden in populations throughout the Indian sub-continent will enable the identification of novel genes associated with many rare and common diseases, potentially facilitating therapeutic development, diagnosis and delivery of precision medicine. The center’s extraordinary high throughput automated gene sequencing and data analysis capabilities present a great opportunity in India and other under-explored populations. According to figures collated by the companies, currently an estimated 90 percent of the potential medicines entering clinical trials fail to demonstrate the necessary efficacy and safety, and never reach patients. Many of these failures are due to incomplete understanding of the link between the biological target of a drug and human disease. By contrast, medicines developed with human genetic evidence have had substantially higher success rates and patient care has benefited.

Global market
The global DNA sequencing market is expected to grow at a CAGR of 20.93 percent during 2018–2022, predicts Research and Markets. One of the major drivers for this market is the all-inclusive cost structure of sequencing products. Manufacturers developed an all-inclusive cost approach that includes the cost of components, direct costs, and indirect costs. This all-inclusive cost structure explains the cost structure to end-users and funding agencies. Also, this approach reduces the volume of sequencing and increase the volume of data. Detailed cost-related data decreases the cost of sequencing and that increases the amount of genome data for better research. Consequently, detailed cost structure is driving the demand for DNA sequencing market.

The latest trend gaining momentum in the market is growth of bioinformatics tools that make DNA sequencing easy. DNA sequencing manufacturers are taking measures to increase the affordability and executability of DNA sequencing. Due to this, the next-generation sequencing market is witnessing the development of cost-effective and time-effective advanced products. The introduction of cost-effective and easy to handle sequencing bioinformatics DNA sequencing tools will further contribute to the market growth. However, one of the major factors hindering the growth of this market is difficulties in clinical interpretations and inadequate reimbursements. Challenges related to clinical samples can affect the growth of the market. A single exome sample can contain up to 20,000 variants, whereas a whole-genome sample will generally have more than three million. Hence, a patient may have to undergo several genetic tests before the disease-causing mutation is identified. This can become expensive and inconvenient for the patient.

The Human Genome Project (HGP) was the first joint initiative by various organizations to map the human genome which took nearly 13 years to complete along with a huge investment. Since the completion of this project, a major transformation has been witnessed in the DNA sequencing technologies. New-age technologies have been developed such as 454 sequencing or Illumina sequencing which involve low cost and deliver results in shorter period of time. Owing to the declining costs of sequencing, many countries are investing in the process which impels the growth prospects of the market. Further, the market is anticipated to be boosted by the rising demand for personalized medicines.

The DNA sequencing market is fragmented owing to the presence of several international and regional vendors. The key players operating in the global DNA sequencing market include Illumina, Thermo Fisher Scientific, Siemens, Pacific Biosciences of California, ZS Genetics, Beckman Coulter, F. Hoffmann-La Roche, Agilent Technologies, Abbott Laboratories, LI-COR, General Electric Company, Myriad Genetics, and Integrated DNA Technologies.

Technological advances
The development of DNA sequencing technologies has a rich history, with multiple paradigm shifts occurring within the last few decades. Current developments in sequencing techniques have dramatically changed the field of genomics during the present technological development, making it promising for even particular research members to generate gigantic amount of sequence data very fast at a considerably subsidized budget and expenses.

High-throughput sequencing. The remarkable advancement of high-throughput sequencing (HTS) technologies has fundamentally changed to understand the genetic and epigenetic molecular bases underlying human health. HTS were developed indispensable for genomic investigation and has been the recent hottest topic for research in the field of genomics, which can generate over 100 times more data in comparison with the most complicated capillary sequencers. Recent advances and developments in HTS using next generation sequencing technologies have become essential in the studies of digital gene expression profiling, in epigenomics, genomics, and transcriptomics. These technologies are dexterous of sequencing multiple DNA molecules in corresponding; and facilitate hundreds of millions of DNA molecules to be sequenced within a short period of time.

Next-generation sequencing. The massively parallel sequencing technology known as next-generation sequencing (NGS) has revolutionized the biological sciences and has been developed in a variety of systems for the investigation of the whole transcriptome for gene expression analysis. It is not an understatement to say that NGS has revolutionized biomedical research. With its ultra-high throughput, scalability, and speed, NGS enables researchers to perform a wide variety of applications and study biological systems at a level never before possible. Today’s complex genomic research questions demand a depth of information beyond the capacity of traditional DNA sequencing technologies. Next-generation sequencing has filled that gap and has become an everyday research tool to address these questions. What once took years of painstaking genetics work – if it could be done at all – can now be performed many times over in the virtual blink of an eye, at less cost and finer resolution, with the ability to process enormous sample sizes and obtain orders of magnitude more data.

Whole exome sequencing. Yet even with the cost of sequencing continuing to drop, it is not always necessary – and is sometimes counterproductive – to sequence an entire genome. Sometimes sequencing a small portion of the genome serves the researcher’s purpose. By using a targeted enrichment strategy, it is possible to pare down the amount of sequencing required for a given project, saving time, sequencing costs, and of course the bioinformatic torrent that comes with all that data. Thus, sequencing only the exome (whole exome sequencing or WES) saves 98–99 percent of the sequencing effort compared with whole genome sequencing (WGS). More targeted sequencing panels can winnow that down even further.

Real-time DNA sequencing. The ability of any lab to sequence the genome of a pathogen quickly and cost-effectively is a game-changer for public health. This is an important tool in the surveillance and control of infectious diseases. It is in the very early days, but real-time DNA sequencing is a significant step forward. It is a step forward for drug developers, too. For biotech, process monitoring for biologics is an obvious application with quantifiable payback. Companies are interested in sequencing to understand antimicrobial resistance, identify airborne pathogens and bioterror threats, and elucidate the relationship between a gene mutation and disease. The combination of speed, portability, and affordability puts real-time WGS within the reach of any scientist in the world. Because the device operates in real time and can be reused, it heralds a new paradigm. There is no need to wait for samples to accumulate for multiplexing, or to send samples to a distant lab for sequencing and analysis. Scientists can run samples cost-effectively when they need them, even if that means running a single sample.

Single-molecule sequencing. Short read massive parallel sequencing has emerged as a standard diagnostic tool in the medical setting. However, short read technologies have inherent limitations such as GC bias, difficulties mapping to repetitive elements, trouble discriminating paralogous sequences, and difficulties in phasing alleles. Long read single molecule sequencers resolve these obstacles. Moreover, they offer higher consensus accuracies and can detect epigenetic modifications from native DNA. The first commercially available long read single molecule platform was the RS system based on single molecule real-time (SMRT) sequencing technology, which has since evolved into their RSII and Sequel systems. Single-molecule sequencing is revolutionizing constitutional, reproductive, cancer, microbial, and viral genetic testing.

Sequencing by expansion. Faster, cheaper DNA sequencing technology has revolutionized medicine in the past 10 years. The technology provides the backbone for breakthroughs in cancer care, personalized treatments, and even wellness plans based on genes. Sequencing by expansion (SBX) is an elegant, low-cost sequencing technology, with a simple workflow and rapid sample-to-measurement process times that can be configured for small, targeted sequence applications as well as large high-throughput whole genome systems. SBX technology encodes the information from a DNA sequence onto another molecule that easier to read. The versatile, low-cost SBX sequencing technology will enable opportunities for a vast array of growing market applications such as in a variety of research-focused industries, including biotechnology, medicine, agriculture, and academia.

Road ahead
DNA sequencing has started making huge strides in the current understanding of mechanisms of various chronic illnesses like cancers, metabolic disorders, inherited disorders, neurodegenerative anomalies, transplant immunology etc. Latest trends in biomedical research are giving birth to a new branch in medicine commonly referred to as precision medicine or personalized therapy. Apart from being a multi-billion dollar a year market, DNA sequencing is one avenue where incremental improvements in research, like discovery of a new gene, for example, can have immediate clinical consequences. While there has been the ability to sequence important parts of the genome, the ability to know the entire sequence has the potential to make huge strides in the area of understanding and predicting disease. The major advances in DNA sequencing technology and its commercial development have driven down the time and cost of sequencing a human genome. In 2018, the industry has at last started to understand the commercial, clinical, regulatory, ethical, and legal issues unlocked by the Human Genome Project.

Increased throughput read length and decreased running cost of current next-generation DNA sequencers is creating an opportunity to obtain high quality, ethnicity specific medical grade genomes that better represents haplotypes common in their regional populations, thereby accelerating the use of precision medicine. India is aggressively gearing up to adopt this current trend and many startups are joining hands to provide clinical diagnosis/prognosis utilizing DNA sequencers. Also there is huge push for HLA donor registries to provide the most compatible match and this is only possible due to the ability of long read DNA sequencers in providing full-length high-resolution allele level typing results and process large sample volumes. Today, the next-generation technology is changing the way doctors manage patients with rare inherited diseases and cancer. It is thus expected that DNA sequencing will continue to improve, with new applications such as screening for disease in newborns in the coming years.

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