Demand for accurate, inexpensive, and fast DNA sequencing data has led to the evolution and dominance of a new generation of sequencing technologies. DNA sequencing contains information about personality traits, genetically determined illnesses, and IQ level of a human being. Therefore, determining legality, ethics, and security concerning control and storage of that data is paramount. The use of genetic information has the potential to provide individuals and medical professionals with an arsenal of life-impacting information. In addition, different sciences are receiving benefits of this technique, including forensic sciences, molecular biology, biotechnology, genetics, anthropology, and archaeology. DNA sequencing is expected to revolutionize the conceptual foundation of these fields.

Since completion of the first human genome sequence, demand for a faster and cheaper sequencing method has increased greatly. This demand has driven the development of second-generation sequencing method/next-generation sequencing (NGS). Despite opening a new edge of genomics research, the fundamental shift away from the conventional sequencing to the NGS technologies has left many applications and capabilities of these new technologies undiscovered, especially those in clinical segment. In addition, the evolution of new sequencing technologies has raised the demand for commercially available platforms, whose diverse applications are restricting the potential of NGS for clinical research and physician scientists.

The current breakthrough in micro fluidics, nanotechnology, imaging, and bioinformatics is expected to provide a novel platform for DNA sequencing market. However, issues related to public health and safety could pose a challenge to the market growth.

Indian Market

The Indian market in 2015 for DNA sequencers is estimated at Rs.175 crore. It may be segmented as capillary sequencers and NGS. The capillary sequencers market is divided in the ratio of 60 percent instruments and 40 percent consumables, whereas the NGS constitutes 70 percent of instruments and 30 percent consumables.

Global Market

The global market for sequencing products has grown to USD 5.9 billion in 2015 from USD 5.3 billion in 2014. The market is expected to grow at a five-year compound annual growth rate (CAGR) of 18.7 percent from 2015 to 2020, reaching nearly USD 13.8 billion by 2020.

In 2015, NGS was the largest workflow segment of the market. The global NGS market was valued at USD 3313.1 million in 2015, estimates Grand View Research. Development of more efficient and rapid genomic sequencing methodologies is anticipated to further drive adoption of NGS platforms consequently influencing industrial growth. HLA testing is anticipated to witness lucrative growth through to 2020 and also account for the largest share in market revenue owing to the application of NGS in deciphering the nucleotide and DNA sequence in the process of HLA typing.

North America was the dominant market for NGS in 2015. This large share can be accounted for by the presence of an enhanced technological healthcare framework, growing adoption of reduction in cost of sequencing per base pair, and high R&D investment for genomic and proteomic sequence determination for biomarkers research. Moreover, majority of the respondents for academic use are located in the U.S., Germany, and U.K. owing to presence of several universities that offer molecular biology courses in this region thereby affecting growth in North American and European NGS industry.

For instance, in June 2015, Illumina Inc. inaugurated its European headquarters in the Cambridge in order to gain a larger share of revenue generated through 10,000 genome projects carried out in the U.K.

Asia-Pacific is expected to grow at the fastest rate with a CAGR of over 18 percent over the next five years owing to the increasing investment for development of healthcare, presence of unmet market demand, growing medical awareness in the regional population, and growing per capita and residual income levels.

Application of these solutions in research projects that are carried out in the universities and research centers is attributive for the largest share of academic research in the market space. Furthermore, scholarships offered for PhD projects using this technology are anticipated to drive the demand for products and services in this industry thereby resulting into lucrative growth through to 2020. Provision of on-site bioinformatics courses that involve the workshops on practical implementation of NGS and data analysis is also expected to boost the revenue generated through academic research in the coming years.

Additionally, presence of clinical research solutions provided by major players like Illumina, Life technologies Corporation, and Agilent Technologies for the purpose of target enrichment and detection has poised this segment to exhibit potentially high-value avenues and opportunities for growth over the next five years.

For example, in June 2015, Life Technologies Corporation's NGS platform based on the principle of ion torrent sequencing was selected for nationwide clinical research program, National Cancer Institute - Molecular Analysis for Therapy Choice (NCI-MATCH) partnership with ECOG-ACRIN.

During 2015, the reagents and consumables segment dominated the market and accounted for more than 57 percent of the market share. Many sequencing suppliers are investing in reagents and consumables, as they generate high ROI due to the presence of a large installed base of equipment. Moreover, many government organizations like the NIH in the U.S. offer grants for quarterly reimbursements for the R&D of consumables, which will contribute to the growth of this market segment in the future.

The global DNA sequencing products market is highly competitive due to the presence of several established vendors that have sizeable market shares. The competition is expected to intensify as vendors are investing in product portfolio expansion and engaging in strategic alliances. Global vendors are increasingly engaging in M&A of local and regional vendors to grow inorganically in the market whereas local and regional vendors are likely to form strategic alliances with global players to enter into the emerging markets and expand their consumer base.

Research Update

An international team of scientists led by Dr Niranjan Nagarajan, from A*STAR's Genome Institute of Singapore (GIS), has released an updated version of GraphMap, a software specifically designed to analyze data from nanopore sequencing, which has been lauded for its potential to revolutionize genomics in being rapid, cheap and portable, and able to provide results in real time. The updated software makes more than 90 percent of the information coming out of Oxford Nanopore Technologies system usable.

The analysis of the DNA code of all life forms has been rapidly advancing due to the availability of new sequencing technologies to read DNA. It may not be too far in the future before multifunction handheld devices can be used to scan, analyze and record data, effectively allowing us to measure and monitor the genetic make-up of daily life. There are currently ongoing efforts to develop such tricorders, first described in the fictional universe of Star Trek, which can sense and intuitively visualize a diverse array of phenomena. For example, saliva and blood samples could be used to diagnose and prevent the spread of infections at home and at work. However, while DNA analysis has become easier, it is still error-prone and could be made more robust. Analytical tools such as GraphMap help compensate for these, using sophisticated algorithms.

Dr Nagarajan, lead author of the study and Principal Investigator of Computational & Systems Biology at the GIS noted, "Advances in DNA technologies have been truly mind-boggling and we are delighted to play a part in this revolution. GraphMap resulted from a wonderful trans-national collaboration with Ivan Sovic and Mile Sikic. Together, we hope that GraphMap will serve as a valuable addition to the toolbox for nanopore sequence analysis."

Race of Scaling

Next-generation sequencing (NGS) systems are evolving rapidly, meaning this should be a core area of focus for a company should they want to excel now and in the near future.

Of the NGS systems, bench-top sequencers have a good niche currently. Although they cannot process as much as the larger high-throughput systems, being smaller has its obvious advantages. In the long term, bench-top sequencers may end up getting squeezed out of their niche, however, finding no room for successful commercialization between the large high-throughput systems and handheld or portable sequencers that are based around nanopore technology.

At this stage, there are several different approaches different companies are working on concerning nanopore-based technologies. Leading the way in this sector of sequencing will certainly help strengthen any company and their competitive position. Many companies are competing, and to a certain extent this is a race of scaling, that is, who can get the most nanopores into the smallest area.

One of the problems that arise from the sequencing process is the flood of data that results. A key issue to note is just how well informatics will be able to handle this data flood. As read lengths become longer and more accurate, the informatics becomes easier. Interpretation of the data is another issue that arises, but focusing on medically actionable determinations should simplify rather than complicate the interpretation of results. Another point to consider is whole genome sequencing, a goal for many. Applications from this process becoming available everywhere at low cost are not too far into the future at the moment.

Next-generation sequencing is an area renowned for its dynamism. Sequencers and their capabilities are in an almost constant state of flux. Continual technological evolution is driving the transfer of sequencing into new environments, while allowing sequencing to become routine in many areas of traditional research.

Sequencing vendors are putting their efforts on improving system performance and extending read lengths. More chemistry kits are available to support target enrichment, an expanding menu of amplicon assays, targeted human DNA sequencing, and targeted RNA sequencing. Furthermore, some vendors are developing novel IT platforms to expand the range of analytical tools available to users.

With all the advantages that potential NGS brings to research and diagnostics, it also has several pitfalls that need to be addressed. The first problem encountered for diagnostics was the massive amounts of data generated. Large-scale sequencing is on the rise with an increasing number of centers lining up to undertake ambitious projects that demand huge volumes of DNA data. Another challenge is the high cost of acquiring equipment, software, and consumables needed for NGS. Analysis and storage of data is another problem faced in the field of data output. The amount of data produced per sequencing cycle on NGS platforms runs into the gigabytes that require specialized high-power computers for quick, effective processing and analysis.

Sequencing is set to become even more accessible, with single molecule real-time sequencing, semi-conductor technology, and simpler chemistry already in use. Nanopore technology is expected to deliver the first fully miniaturized system, as well as the next big leap in sequencing performance and cost. These exciting advances suggest that the current trajectory of technological progress in DNA sequencing is likely to continue, extending the sequencing revolution from the lab into the clinic.

Dr Pravin D Potdar, Head-Department of Molecular Medicine & Biology, Jaslok Hospital & Research Centre, Mumbai
Second Opinion
NGS in Cancer Diagnosis and Therapies

The latest innovations in DNA technology have significantly influenced the diagnosis and therapies of cancer. In the year 2003, Francis Collins published the complete Genome Project. Thereafter, there has been advancement in the field of genomics which has led to better understanding of cancer biology and also helped in the development of various approaches to manage treatment of various cancers. Recently the introduction of next-generation sequencing (NGS)-based technologies has revolutionized the field of cancer diagnosis and therapies. NGS technology has an added advantage that the massive number of reactions can be uniformly and accurately sequenced in a high-throughput manner. The traditional Sanger sequencing required decades for whole genome sequencing whereas using NGS, it is now possible to accomplish such feat in a matter of days at the expense of both reduction in cost and time. The NGS market as of 2016 is USD 4031 million and is expected to reach USD 10,371 million by 2021.

Indian market has also started to invest in NGS platform and is expected to witness a boost in demand. Various bench-top NGS platforms have been developed by different companies to provide high-quality data for both research and clinical applications. The second-generation systems such as Illumina MiSeq & HiSeq, 454 Pyrosequencing, and Ion Torrent utilize different sequencing chemistries of immobilized amplified DNA-molecules. They not only help in deciphering all the spectrum of cancer-associated hereditary mutations in genes such as EGFR, TP53, BRAF, KRAS, PTEN, BRCA1, and BRCA2 but also provide the complete molecular profiling of tumor in a single assay. Thus, it offers a simple workflow for clinical laboratories to process hundreds of samples and quick delivery of answers at the same time with digital accuracy. NGS also offers both specificity and sensitivity to detect circulating tumor DNA (ctDNA) from liquid biopsies in noninvasive approach for early detection of cancer. The third-generation system (Pacific Biosciences, Oxford Nanopore) also known as single-molecule sequencing avoids the biased clonal amplification but generates long kb-sized reads and also detects the epigenetic lesions associated with cancer in real time. In the near future, every patient will have their own personal genome sequenced, which will enable molecular subtyping of disease with the rational use of molecular-guided therapies.

Dr Pravin D Potdar
Head-Department of Molecular Medicine & Biology,
Jaslok Hospital & Research Centre,

Nagendra Kumar Singh, National Professor BP Pal Chair, National Research Centre on Plant Biotechnology, Pusa Campus, New Delhi
Second Opinion
Making Decoding Faster and Affordable

Initially, the sequencing was slow and cumbersome because of use of radioactivity and manual detection of sequences on X-ray films. Sanger's method was slowly automated during 1990s with the use of fluorescence-based automated detection of DNA sequence ladders, a development that made possible the decoding of large model eukaryotic genomes at the turn of this century. Major revolution in DNA sequencing technologies was achieved in 2008 when three independent approaches were used to carry out massive parallel sequencing of DNA, reducing the cost of sequencing and increasing the speed of sequencing at least by two orders of magnitudes.

These technologies did away with the need for cloning DNA fragments in bacteria for sequencing. Instead, they relied on PCR amplification of DNA fragments to prepare library for sequencing. However, two of these technologies (~50 bp in SOLiD by ABI) and ~100 bp in Illumina by Solexa have the disadvantage of short read lengths, which create difficulty in assembly of large genomes with repetitive sequences, although 454 pyrosequencing by Roche has relative longer read lengths of over 500 bp and had clear advantage in genome assembly but per base sequencing cost was higher with this. Now we have entered the era of third-generation sequencing technologies, which do not require even PCR amplification.

These are single-molecule real-time DNA sequencing offered by PacBio (already a commercial success) and Oxford Nanopore system (still under trial). Single-molecule sequencing offers read lengths in excess of 10,000 bp and also allows analysis of DNA modifications as DNA is analyzed in real time in its original form. Similarly, single-molecule-based DNA fingerprinting techniques have also become available to help genome assemblies.

Due to its very high throughput and low cost of sequencing per base pair, Illumina has edge for re-sequencing and detection of variation as well as genotyping by sequencing, while PacBio has an edge in assembly of large genomes without sequence gaps. Ion Torrent PGM machines, which are actually improved and cost-effective version of Roche 454 technology, are suitable for medium throughput targeting re-sequencing. These developments in sequencing technologies offer a wide option for cost-effective sequencing technologies for genome assembly and genotyping.

Nagendra Kumar Singh
National Professor BP Pal Chair,
National Research Centre on Plant Biotechnology, Pusa Campus,
New Delhi

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