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Miniaturized Nanopore DNA Sequencing: Accelerating the Path to Precision Medicine.

On January 30,2015, President Barack Obama announced the launch of a Precision Medicine Initiative--"a bold new research effort to revolutionize how we improve health and treat disease" (1). The success of this bold new initiative depends upon continued improvements in the genomics space. Arguably, the key technological player in precision medicine is next generation sequencing (NGS), [2] an extremely powerful technology for detection of genetic variation. NGS has enabled a multitude of impressive research discoveries that have been translated toward more rapid, cost-effective, and comprehensive analyses for inherited conditions and cancer. However, despite its achievements, NGS analysis is still too expensive and time-consuming of a process to be carried out routinely on all patients. The technological platform and the clinical-grade reagents themselves are both costly, as is the scientific know-how to perform the benchwork, bioinformatics, and variant interpretation. Reducing the time, cost, and effort to perform clinical NGS is essential to achieve further adoption of personalized medicine.

Current NGS sequencing technologies generally use expensive and slow optic-based measurements of short copies of PCR-amplified DNA. Additionally, 3 major groups of genomic variation that are important for clinical diagnostics cannot be efficiently detected by de novo assembly of sequencing data from short DNA strands. The first of these, and closest to clinical optimization, are the copy number alterations that remove or add single or multiple exons, such as those commonly encountered in Duchene muscular dystrophy. The next major class is the trinucleotide repeat disorders. The classical example here is Fragile X, whereby a repeating CGG motif expansion (growing to over thousands of nucleotides in some cases) causes disease. Finally, regions of low sequence complexity, known as pseudogenes, or highly related gene family members, like members of the CYP family of genes, can be challenging to detect a real pathogenic change from one that is in the nonexpressed copy, or accurately gauge the total number of active vs inactive copies of the gene. Each of these types of alterations are quite common in genetic disease, yet cannot be routinely detected by current NGS methodologies.

A newer technology, nanopore-based sequencing, with its ability to sequence long single strands of DNA, is being hailed for its potential to realize the dream of ultrafast and inexpensive genome sequencing, overcoming many of the technical and variant-detection limitations of traditional NGS technologies. Nanopore-based DNA sequencing has been explored since the 1990s, when it was discovered that single-stranded DNA could be driven across a lipid bilayer through [alpha]-hemolysin pores (2). In 2014, Oxford Nanopore Technologies unveiled its commercial portable MinION nanopore sequencing instrument, which is a small, mobile phone--sized USB device, but it currently has an error rate of around 15% (3). Despite this obvious shortcoming, there has been a lot of excitement around the potential of this new approach to DNA sequencing, with several different types of nanopore-based sequencing methods now being explored.

The benefits to the described single-molecule sequencing platform are numerous. First, amplification bias, a common issue in traditional NGS sequencers, can be eliminated through single-molecule sequencing. This is important because there are many GC-rich regions of the genome that create amplification bias and cannot be effectively interrogated with traditional NGS methods, thus requiring the use of supplementary methods. Adding these supplementary methods increases the cost of the test and complexity of the workflow. Second, the electronic chip platform enables a much more high-throughput approach compared to current methods, thereby greatly increasing efficiency and speed of turn-around. Third, it is anticipated that long reads produced by this technology will greatly enhance the ability to address the aforementioned challenging genomic scenarios like copy number variants (CNVs) and trinucleotide repeat expansions as well as provide haplotype information and identify alternatively spliced mRNAs.

Recently, details of a proof of principle study collaboration between Genia (acquired by Roche in 2014) and Jingyue Ju's group at Columbia University involving the construction of a new type of miniaturized nanopore-based single-molecule sequencer were revealed (4). Ju's approach uses sequencing by synthesis (SBS), where sequential tagged nucleotides are detected in nanopores during enzyme-catalyzed DNA synthesis (Fig. 1). As each tagged nucleotide translocates through the pore, ionic current blockades are produced that reflect the chemical structure of the tagged nucleotide. Each polymer tag produces a distinct ionic current blockade signal, enabling real-time, single base resolution. The polymer tags on each nucleotide provide an advantage over previous nanopore sequencing methods because the more distinct chemical structure of the tags (as compared to the highly similar chemical structure of nucleotides) in theory creates a more specific and accurate test. Ju's group demonstrated accurate sequence of 20 bases in their published work.

As with most innovations in the DNA sequencing field, early proof of concept experiments generate a lot of promise and speculation. To convert that into realized benefits, especially in the environment of clinical diagnostics, the work by Ju's group will have to address some key limitations. Much of the hype over this technology is its ability to produce longer reads, which will greatly advance our current technology's ability to identify haplotypes and detect CNVs, trinucleotide repeats, and low complexity regions. However, accurate reads of only 20 nucleotides, as described in the publication, are not suitable for robust detection of insertions and deletions, and is quite far behind the current state of the art NGS instruments. Although the data have not yet been published, Ju has indicated that they are now able to achieve read lengths of 1000 bases, although accuracy is yet to be determined (5).

As described above, nanopore sequencing is advantageous because of its rapid sequencing ability, and the system described by Ju's group is very fast. In fact, to optimize the speed of tag detection, the authors chose to develop a system that would monitor the blockade current of the tags while they were still attached to the nucleotides. In this system, detection of the tag occurs before nucleotide incorporation by phosphoryl transfer, rather than after the tags are detached from their nucleotides. However, too much speed in this system can be disadvantageous from an accuracy standpoint, and the authors have encountered instances of multiple signals being generated from a single SBS event (referred to as "stuttering"). In the diagnostic world, this could lead to the false positive assertion that there is an insertional (likely frameshifting) event, creating a potentially erroneous diagnosis. This type of event also does nothing to improve upon the already problematic inaccurate calls in homopolymer regions with the current NGS technologies. To address the issue with stuttering, the authors were able to change the chemistry of the reaction to slow and stabilize the process (and they also ignored dwell times <10 ms), but without documented data on what limitations this change would introduce to the system.

Another potential hurdle to overcome with the nanopore SBS technology is in the creation of the reagents and critical components of the assay. For example, building the tagged nucleotides seems problematic, and there are several issues with assembling the nanopores and inserting them into the lipid membrane. The durability of bionanopores with lipid membranes is also questionable since they may denature at high temperature and high salt concentrations. Solid-state nanopores, such as those manufactured with silicon nanofabrication technology, may solve many of these issues, but require further exploration. Taken together, overcoming these chemistry hurdles could lead to a very expensive suite of reagents needed to power the sequencing system. Again, this would be a major impediment to successful adoption since the triad of requirements includes speed, accuracy, and cost.

While the development of an inexpensive/rapid nanopore sequencer has generated a lot of excitement, there is still much work that needs to be done to make a commercially viable diagnostic-grade product. This work involves optimizing parameters, including increased resolution of blockade signatures and improved reaction conditions to minimize stuttering (e.g., by adjusting the relative concentration of catalytic and noncatalytic metal cofactors). Ju and colleagues also point out that they would like to construct integrated circuits that include many more electrodes with faster measurement capabilities (4). Furthermore, it must be acknowledged that only a fraction of the true cost of DNA sequencing is in the sequencing itself. Translating DNA base changes into knowledge for efficient and informed patient reporting is equally in need of revolutionizing. Therefore, it should be noted that while each new and improved iteration of nanopore sequencing is another (important) technological step, the proof of the transformative nature of this technology remains to be seen.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest.

References

(1.) The White House, Office of the Press Secretary. Remarks by the President on precision medicine.https://www. whitehouse.gov/the-press-office/2015/01/30/remarkspresident-precision-medicine (Accessed September 2016).

(2.) KasianowiczJJ, Brandin E, Branton D, Deamer DW. Characterization of individual polynucleotide molecules using a membrane channel. Proc Natl Acad Sci 1996;93:13770 -3.

(3.) Jain M, Fiddles IT, Miga KH, Olsen HE, Paten B, Akeson M. Improved data analysis for the MinlON nanopore sequencer. Nat Meth 2015;12:351-6.

(4.) Fuller CW, Kumar S, Mintu P, Chien M, Bibillo A, Stranges PB, et al. Real-time single-molecular electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array. Proc Natl Acad Sci 2016;113:5233-8.

(5.) Columbia Engineering, The Fu Foundation School of Engineering and Applied Science. Columbia Engineeringled team advances single molecule electronic DNA sequencing-a future platform for precision medicine. http:// engineering.columbia.edu/columbia-engineering-ledteam-advances-single-molecule-electronic-dna-sequencing %E2%80%94future-platform-0 (Accessed January 2017).

Linnea M. Baudhuin [1] * ([dagger]) and Matthew J. Ferber [1] * ([dagger])

[1] Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN.

[2] Nonstandard abbreviations: NGS, next generation sequencing; CNV, copy number variant; SBS, sequencing by synthesis.

* Address correspondence to: L.M.B. at Department of Laboratory Medicine and Pathology, Mayo Clinic, 200 First St. SW, Rochester, MN, 55905. Fax 507-284-9758; e-mail baudhuin.linnea@mayo.edu. M.J.F. at Department of Laboratory Medicine and Pathology, Mayo Clinic, 200 First St. SW, Rochester, MN, 55905. Fax 507-266-4176; e-mail ferber.matthew@mayo.edu.

([dagger]) Linnea M. Baudhuin and Matthew J. Ferber contributed equally to the work, and both should be considered as first authors.

Received August 3, 2016; accepted September 8, 2016.

Previously published online at DOI: 10.1373/clinchem.2016.261420

Caption: Fig. 1. Schematic of nanopore SBS.

An [Alpha]-hemolysin nanopore heptamer and single DNA polymerase molecule are covalently attached together. The incorporating nucleotide is tagged with a structurally distinct polymer. As DNA synthesis takes place, the polymer end of the tagged nucleotide enters the hemolysin nanopore opening, where a distinct current blockade is produced and measured. Following completion of the polymerase catalytic step, the polymer tag is released and translocates through the pore, thereby ending the blockade signal. Used with permission of the Mayo Foundation for Medical Education and Research. All rights reserved.
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Title Annotation:Perspective
Author:Baudhuin, Linnea M.; Ferber, Matthew J.
Publication:Clinical Chemistry
Date:Mar 1, 2017
Words:1857
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