February 15, 2018 (Vol. 38, No. 4)

Randi Hernandez Managing Editor GEN

An Orthogonal Approach to Genome Sequencing

(Part I of a two-part series)

Intense competition in the genomic-sequencing space continues, but not at a level that once fueled the field. Could progress in sequencing tools and research overseas help democratization of sequencing in the United States?

The $1,000 genome—or even the $100 genome—could be closer to becoming a reality than ever before. Thanks in large part to the prowess of Illumina, the cost of DNA sequencing has decreased by four orders of magnitude during the period 2007–2012.1

But, in the past few years, the rate at which the cost of sequencing has been dropping has plateaued. Competition has also slowed, experts say.1,2

For the past decade, since the acquisition of the British firm Solexa in 2006, Illumina has been the dominant player in the market. Illumina claims to be the platform that has delivered the $1,000 genome and produced more than 90% of the world’s genome sequence data. Over the years, Illumina has fought off competition from Life Technologies and Ion Torrent (Thermo Fisher Scientific), 454 Life Sciences (Roche), and other companies with sequencing systems, including those developed by Complete Genomics and Helicos BioSciences. Some of these technologies are off the market or have been gobbled up by competitors. But two platforms that feature longer individual reads—sequencers from Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT)—offer researchers some important advantages, particularly for work on complex genomes.

“It’s hard to say who Illumina’s ‘fiercest’ competitor is because Illumina is so dominant,” observed Shawn Baker, a genomics advisor and consultant at SanDiegOmics (shawn@sandiegomics.com). “They have lots of competition, but each competitor only competes in certain niches—nobody covers the entire sequencing space like Illumina.” Baker said if he had to choose, Ilumina’s biggest competitor is probably Thermo Fisher, although “PacBio, ONT, and BGI are also competitors in certain markets.”

Investigators in the United Kingdom achieved the first assembly of a 30× (30-fold coverage) human genome using primarily ONT long-read data (according to an article published in Nature Biotechnology).3 A tweet on January 12, 2018 from ONT’s chief technology officer, Clive Brown, announced the achievement of a milestone using its PromethION instrument: runs equivalent to 30× coverage (human) for less than $1,000.4

As Brown told GEN exclusively: “The market leader, Illumina, will no longer have the only shippable instrument system capable of sub-$1,000 human genome sequencing. This will be the beginning of a potential threat to their market dominance.”

Are We There Yet?

Many commentators (including GEN contributor Kevin Davies, Ph.D., in his book, The $1,000 Genome) have predicted that the $1,000 genome would eventually become a reality. Illumina Executive Chairman Jay Flatley predicted Illumina would cross that threshold a few years ago. But it’s 2018, and whether the field is truly there depends on how you do the math.

Baker thinks that Illumina’s NovaSeq S4 flow cell—one of the company’s newer models—will enable users to generate whole human genomes for approximately $800. Illumina’s Director of Product Marketing Joel Fellis, Ph.D., told GEN at the American Society of Human Genetics (ASHG) conference in 2017 that Illumina sees a path to the $100 genome, but did not offer details about how (or when) the company would achieve that major milestone.

Dr. Fellis admitted that for labs not operating NovaSeq at full capacity, the cost of reagents alone could easily be $800.5 So, for labs not running the NovaSeq at full capacity, the $1,000 genome is not as realistic as for research centers operating at scale.

Illumina’s top-of-the-line platform, the NovaSeq 6000 System, costs almost $1 million. Despite that hefty price, the company says its newest products that support the NovaSeq technology pave the way for “large-population-scale initiatives at the lowest price per sample.”6 Dr. Fellis noted that one-third of Illumina’s customers are new and estimated that 90% of the sequencing data that exist have been generated using Illumina’s machines.

Costs aside, there is plenty of evidence supporting the clinical utility of sequencing. Many recent studies have demonstrated that incorporating sequencing equipment at the research level will, in fact, have a major impact on clinical decision-making. And research-driven genomics will eventually transform into healthcare-driven calculations.

One study, which was conducted by Birney et al.,7 asserted that genomic analysis would have the most impact in certain disease states, including infectious disease, rare disease, cancer, and common or chronic disease. In fact, according to this study, genomics will be used in 70% of cancer diagnoses by 2027—unless the cost of sequencing plummets further, in which case, genomics will likely play a pivotal role in oncology prior to 2027.

The Long and Short of It

A lab’s sequencing strategy depends on a handful of factors. For example, does the research team have a good idea about where a mutation is likely to occur or have a specific gene variant in mind to examine? If so, short-read sequencing—individual reads of 50–250 bases—is likely ideal. Most human exome- and whole-genome sequencing (WGS) is performed using Illumina’s short-read platform.

If researchers are interested in assembling the whole genome of an organism de novo—sequencing DNA that is highly repetitive or obtaining haplotype information—long reads are much preferred.

Cost may also be a consideration when investigators are thinking about their sequencing needs. According to Andy Felton, Ph.D., head of product development at Ion Torrent (a Thermo Fisher Scientific product), reagent costs rise when you go from looking at exomes to WGS. He told GEN at ASHG 2017 that restricting a project’s scope to targeted sequences cuts down on complexity and cost.

Whether looking at the whole genome or just parts of it, experts argue that once long reads come down in price, short reads won’t even be used—at least not as commonly as they are today.

“There are claims of which is better, short or long, but ultimately, conflicts of interest arise among providers that do their own analyses on sequencing performance,” says Vural Özdemir, M.D., Ph.D., editor-in-chief of the journal OMICS: A Journal of Integrative Biology.

Most research scientists, in principle, might be interested in long reads. “But if you have to take a stance,” maintains Dr. Özdemir, “I would opt for the short reads at the moment, both for cost and clinical utility.”

As mentioned, clinical labs need throughput and accuracy, and have historically relied on Illumina (and a bit of traditional Sanger sequencing for confirmation). But WGS is not always necessary to gain useful information about a disease. Looking at only parts of the genome through short reads can be informative, as well.

Short Reads: Are They Still Worth the Time?

When sequencing focuses on known genes in a disease area, short reads are a preferable method of analysis. According to Pan Zhang, Ph.D., M.D., director of the Sequencing and Microarray Center at the Coriell Institute for Medical Research, “Genome-wide associated studies have not proven fruitful for hearing-loss research. For this poorly understood genetic condition, you need a discovery tool that is both rapid and cost-effective for analyzing multiple genes in depth in many samples,” said Dr. Zhang in a Thermo press release.8

Thermo is leveraging its tool as one that is specifically tailored to less data-intensive applications, and for identifying targeted sequencing in a clinical setting, such as how it was used in the aforementioned hearing-loss study. Thermo is taking this stance mostly because, according to Baker, “the platform was never able to scale up enough to compete with Illumina’s larger instruments.” Baker adds that the Ion Torrent platform has a higher error rate and a known difficulty with homomers.

David Smith, M.D., director of the Mayo Clinic’s Technology Assessment Group at the Center for Individualized Medicine, says that while widely used, short reads have two major disadvantages: sequence accuracy is not great, and the technique produces a fraction of output that has to be assembled after multiple reads.

1. J. Shendure et al., “DNA Sequencing at 40: Past, Present and Future,” Nature 550, 345–353 (October 19, 2017), doi:10.1038/nature24286.
2. National Human Genome Research Institute, “DNA Sequencing Costs: Data from the NHGRI Genome Sequencing Program (GSP),” accessed January 19, 2018.
3. M. Jain et al., “Nanopore Sequencing and Assembly of a Human Genome with Ultra-Long Reads,” Nat. Biotechnol. (published online January 29, 2018), doi: 10.1038/nbt.4060.
4. Tweet from Clive Brown (@Clive_G_Brown), accessed on January 19, 2018.
5. National Human Genome Research Institute, “The Cost of Sequencing the Human Genome,” accessed January 19, 2018.
6. Press Release, “Illumina Releases NovaSeq S4 Flow Cell and NovaSeq Xp Workflow,” accessed January 19, 2018.
7. E. Birney et al., “Genomics in Healthcare: GA4GH Looks to 2022,” bioRxiv (bioRxiv preprint first posted online October 15, 2017).
8. Thermo Fisher Scientific, Press Release, “Thermo Fisher Scientific Customers to Showcase Innovations in Precision Genomics Research for Inherited Disease and Reproductive Health at ASHG,” accessed January 22, 2018.
9. BGI, Press Release, “BGI’s MGI Tech Launches Two New NGS Platforms,” (October 31, 2017), accessed January 22, 2018.
10. Thermo Fisher Scientific, “Thermo Fisher Scientific Introduces Ion GeneStudio S5 Series, A Line of Highly Versatile Next Generation Sequencers,” accessed January 22, 2018.
11. Stratos Genomics, Press Release, “Stratos Genomics Raises Funds to Ready for Commercialization,” accessed January 22, 2018.
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13. S. Goodwin, J.D. McPherson, and W.R. McCombie, “Coming of Age: Ten Years of Next-Generation Sequencing Technologies,” Nat. Rev. Genet.17(6), 333–351 (May 17, 2016), doi: 10.1038/nrg.2016.49.
14. M.J.P. Chaisson et al., “Multi-Platform Discovery of Haplotype-Resolved Structural Variation in Human Genomes,” bioRxiv, (bioRxiv preprint first posted online September 23, 2017).
15. Y. Mostovoy et al., “A Hybrid Approach for De Novo Human Genome Sequence Assembly and Phasing,” Nat. Methods 13, 587–590 (2016), doi:10.1038/nmeth.3865.
16. J. Quick et al., “Multiplex PCR method for MinION and Illumina Sequencing of Zika and Other Virus Genomes Directly from Clinical Samples,” Nat. Protocols 12, 1261–1276 (2017), doi:10.1038/nprot.2017.066.
17. Pacific Biosciences, Press Release, “BGI Increases Long-Read Sequencing Capacity with Purchase of 10 PacBio Sequel Systems,” accessed January 25, 2018.
18. Illumina, Press Release, “Thermo Fisher Scientific and Illumina Sign Agreement to Provide Research Market Broader Access to Ion AmpliSeq Technology,” accessed January 22, 2018.

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