The ASBMB story notes that nanopore technology depends on differences in charges on either side of the membrane to force DNA or RNA through the hole. This is one reason why proteins pose such a challenge.
“Think of a cell as a miniature city, with proteins as its inhabitants. Each protein-resident has a unique identity, its own characteristics, and function. If there was a database cataloging the fingerprints, job profiles, and talents of the city’s inhabitants, such a database would undoubtedly be invaluable!” said Behzad Mehrafrooz, PhD (above), Graduate Research Assistant at University of Illinois at Urbana-Champaign in an article he penned for the university website. This research should be of interest to the many clinical laboratories that do protein testing. (Photo copyright: University of Illinois.)
How the Maglia Process Works
In a Groningen University news story, Maglia said protein is “like cooked spaghetti. These long strands want to be disorganized. They do not want to be pushed through this tiny hole.”
His technique, developed in collaboration with researchers at the University of Rome Tor Vergata, uses electrically charged ions to drag the protein through the hole.
“We didn’t know whether the flow would be strong enough,” Maglia stated in the news story. “Furthermore, these ions want to move both ways, but by attaching a lot of charge on the nanopore itself, we were able to make it directional.”
The researchers tested the technology on what Maglia described as a “difficult protein” with many negative charges that would tend to make it resistant to flow.
“Previously, only easy-to-thread proteins were analyzed,” he said in the news story. “But we gave ourselves one of the most difficult proteins as a test. And it worked!”
Maglia now says that he intends to commercialize the technology through a new startup called Portal Biotech.
Detecting Post-Translational Modifications in the UK
In another recent study, researchers at the University of Oxford reported that they have adapted nanopore technology to detect post-translational modifications (PTMs) in protein chains. The term refers to changes made to proteins after they have been transcribed from DNA, explained an Oxford news story.
“The ability to pinpoint and identify post-translational modifications and other protein variations at the single-molecule level holds immense promise for advancing our understanding of cellular functions and molecular interactions,” said contributing author Hagan Bayley, PhD, Professor of Chemical Biology at University of Oxford, in the news story. “It may also open new avenues for personalized medicine, diagnostics, and therapeutic interventions.”
Bayley is the founder of Oxford Nanopore Technologies, a genetic sequencing company in the UK that develops and markets nanopore sequencing products.
The news story notes that the new technique could be integrated into existing nanopore sequencing devices. “This could facilitate point-of-care diagnostics, enabling the personalized detection of specific protein variants associated with diseases including cancer and neurodegenerative disorders,” the story states.
In another recent study, researchers at the University of Washington reported that they have developed their own method for protein sequencing with nanopore technology.
“This opens up the possibility for barcode sequencing at the protein level for highly multiplexed assays, PTM monitoring, and protein identification!” Motone wrote.
Single-cell proteomics, enabled by nanopore protein sequencing technology, “could provide higher sensitivity and wider throughput, digital quantification, and novel data modalities compared to the current gold standard of protein MS [mass spectrometry],” they wrote. “The accessibility of these tools to a broader range of researchers and clinicians is also expected to increase with simpler instrumentation, less expertise needed, and lower costs.”
There are approximately 20,000 human genes. However, there are many more proteins. Thus, there is strong interest in understanding the human proteome and the role it plays in health and disease.
Technology that makes protein testing faster, more accurate, and less costly—especially with a handheld analyzer—would be a boon to the study of proteomics. And it would give clinical laboratories new diagnostic tools and bring some of that testing to point-of-care settings like doctor’s offices.
And in less than eight hours, they had diagnosed a child with a rare genetic disorder, results that would take clinical laboratory testing weeks to return, demonstrating the clinical value of the genomic process
In another major genetic sequencing advancement, scientists at Stanford University School of Medicine have developed a method for rapid sequencing of patients’ whole human genome in as little as five hours. And the researchers used their breakthrough to diagnose rare genetic diseases in under eight hours, according to a Stanford Medicine news release. Their new “ultra-rapid genome sequencing approach” could lead to significantly faster diagnostics and improved clinical laboratory treatments for cancer and other diseases.
“A few weeks is what most clinicians call ‘rapid’ when it comes to sequencing a patient’s genome and returning results,” said cardiovascular disease specialist Euan Ashley, MD, PhD (above), professor of medicine, genetics, and biomedical data science, at Stanford University in the news release. “The right people suddenly came together to achieve something amazing. We really felt like we were approaching a new frontier.” Their results could lead to faster diagnostics and clinical laboratory treatments. (Photo copyright: Stanford Medicine.)
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Need for Fast Genetic Diagnosis
In their NEJM paper, the Stanford scientists argue that rapid genetic diagnosis is key to clinical management, improved prognosis, and critical care cost savings.
“Although most critical care decisions must be made in hours, traditional testing requires weeks and rapid testing requires days. We have found that nanopore genome sequencing can accurately and rapidly provide genetic diagnoses,” the authors wrote.
To complete their study, the researchers sequenced the genomes of 12 patients from two hospitals in Stanford, Calif. They used nanopore genome sequencing, cloud computing-based bioinformatics, and a “custom variant prioritization.”
Their findings included:
Five people received a genetic diagnosis from the sequencing information in about eight hours.
Diagnostic rate of 42%, about 12% higher than the average rate for diagnosis of genetic disorders (the researchers noted that not all conditions are genetically based and appropriate for sequencing).
Five hours and two minutes to sequence a patient’s genome in one case.
Seven hours and 18 minutes to sequence and diagnose that case.
How the Nanopore Process Works
To advance sequencing speed, the researchers used equipment by Oxford Nanopore Technologies with 48 sequencing units called “flow cells”—enough to sequence a person’s whole genome at one time.
The Oxford Nanopore PromethION Flow Cell generates more than 100 gigabases of data per hour, AI Time Journal reported. The team used a cloud-based storage system to enable computational power for real-time analysis of the data. AI algorithms scanned the genetic code for errors and compared the patients’ gene variants to variants associated with diseases found in research data, Stanford explained.
According to an NVIDIA blog post, “The researchers accelerated both base calling and variant calling using NVIDIA GPUs on Google Cloud. Variant calling, the process of identifying the millions of variants in a genome, was also sped up with NVIDIA Clara Parabricks, a computational genomics application framework.”
Rapid Genetic Test Produces Clinical Benefits
“Together with our collaborators and some of the world’s leaders in genomics, we were able to develop a rapid sequencing analysis workflow that has already shown tangible clinical benefits,” said Mehrzad Samadi, PhD, NVIDIA Senior Engineering Manager and co-author of the NEJM paper, in the blog post. “These are the kinds of high-impact problems we live to solve.”
In their paper, the Stanford researchers described their use of the rapid genetic test to diagnose and treat an infant who was experiencing epileptic seizures on arrival to Stanford’s pediatric emergency department. In just eight hours, their diagnostic test found that the infant’s convulsions were attributed to a mutation in the gene CSNK2B, “a variant and gene known to cause a neurodevelopmental disorder with early-onset epilepsy,” the researchers wrote.
“By accelerating every step of this process—from collecting a blood sample to sequencing the whole genome to identifying variants linked to diseases—[the Stanford] research team took just hours to find a pathogenic variant and make a definitive diagnosis in a three-month-old infant with a rare seizure-causing genetic disorder. A traditional gene panel analysis ordered at the same time took two weeks to return results,” AI Time Journal reported.
New Benchmarks
The Stanford research team wants to cut the sequencing time in half. But for now, the five-hour rapid whole genome sequence can be considered by clinical laboratory leaders, pathologists, and research scientists a new benchmark in genetic sequencing for diagnostic purposes.
Stories like Stanford’s rapid diagnosis of the three-month old patient with epileptic seizures, point to the ultimate value of advances in genomic sequencing technologies.
With 100% of the human genome mapped, new genetic diagnostic and disease screening tests may soon be available for clinical laboratories and pathology groups
Utilizing technology developed by two different biotechnology/genetic sequencing companies, an international consortium of genetic scientists claim to have sequenced 100% of the entire human genome, “including the missing parts,” STAT reported. This will give clinical laboratories access to the complete 3.055 billion base pair (bp) sequence of the human genome.
If validated, this achievement could greatly impact future genetic research and genetic diagnostics development. That also will be true for precision medicine and disease-screening testing.
Completing the First “End-to-End” Genetic Sequencing
In June of 2000, the Human Genome Project (HGP) announced it had successfully created the first “working draft” of the human genome. But according to the National Human Genome Research Institute (NHGRI), the draft did not include 100% of the human genome. It “consists of overlapping fragments covering 97% of the human genome, of which sequence has already been assembled for approximately 85% of the genome,” an NHGRI press release noted.
“The original genome papers were carefully worded because they did not sequence every DNA molecule from one end to the other,” Ewan Birney, PhD, Deputy Director General of the European Molecular Biology Laboratory (EMBL) and Director of EMBL’s European Bioinformatics Institute (EMBL-EBI), told STAT. “What this group has done is show that they can do it end-to-end. That’s important for future research because it shows what is possible,” he added.
In their published paper, the T2T scientists wrote, “Addressing this remaining 8% of the genome, the Telomere-to-Telomere (T2T) Consortium has finished the first truly complete 3.055 billion base pair (bp) sequence of a human genome, representing the largest improvement to the human reference genome since its initial release.”
Tale of Two Genetic Sequencing Technologies
Humans have a total of 46 chromosomes in 23 pairs that represent tens of thousands of individual genes. Each individual gene consists of numbers of base pairs and there are billions of these base pairs within the human genome. In 2000, scientists estimated that humans have only 30,000 to 35,000 genes, but that number has since been reduced to just above 20,000 genes.
According to STAT, “The work was possible because the Oxford Nanopore and PacBio technologies do not cut the DNA up into tiny puzzle pieces.”
PacBio used HiFi sequencing, which is only a few years old and provides the benefits of both short and long reads. STAT noted that PacBio’s technology “uses lasers to examine the same sequence of DNA again and again, creating a readout that can be highly accurate.” According to the company’s website, “HiFi reads are produced by calling consensus from subreads generated by multiple passes of the enzyme around a circularized template. This results in a HiFi read that is both long and accurate.”
Oxford Nanopore uses electrical current in its sequencing devices. In this technology, strands of base pairs are pressed through a microscopic nanopore one molecule at a time. Those molecules are then zapped with electrical currents to enable scientists to determine what type of molecule they are and, in turn, identify the full strand.
The T2T Consortium acknowledge in their paper that they had trouble with approximately 0.3% of the genome, but that, though there may be a few errors, there are no gaps.
“You’re just trying to dig into this final unknown of the human genome,” Karen Miga (above), Assistant Professor in the Biomolecular Engineering Department at the University of California, Santa Cruz (UCSC), Associate Director at the UCSC Genomics Institute, and lead author of the T2T Consortium study, told STAT. “It’s just never been done before and the reason it hasn’t been done before is because it’s hard.” (Photo copyright: University of California, Santa Cruz.)
Might New Precision Medicine Therapies Come from T2T Consortium’s Research?
The researchers claim in their paper that the number of known base pairs has grown from 2.92 billion to 3.05 billion and that the number of known genes has increased by 0.4%. Through their research, they also discovered 115 new genes that code for proteins.
The T2T Consortium scientists also noted that the genome they sequenced for their research did not come from a person but rather from a hydatidiform mole, a rare growth that occasionally forms on the inside of a women’s uterus. The hydatidiform occurs when a sperm fertilizes an egg that has no nucleus. As a result, the cells examined for the T2T study contained only 23 chromosomes instead of the full 46 found in most humans.
Although the T2T Consortium’s work is a huge leap forward in the study of the human genome, more research is needed. The consortium plans to publish its findings in a peer-reviewed medical journal. In addition, both PacBio and Oxford Nanopore plan to develop a way to sequence the entire 46 chromosome human genome in the future.
The future of genetic research and gene sequencing is to create technologies that will allow researchers to identify single nucleotide polymorphisms (SNPs) that contain longer strings of DNA. Because these SNPs in the human genome correlate with medical conditions and response to specific genetic therapies, advancing knowledge of the genome can ultimately provide beneficial insights that may lead to new genetic tests for medical diagnoses and help medical professionals determine the best, personalized therapies for individual patients.
Medical laboratories are already using gene sequencing as part of a global effort to identify new variants of the coronavirus and their genetic ancestors
Thanks to advances in genetic sequencing technology that enable medical laboratories to sequence organisms faster, more accurately, and at lower cost than ever before, clinical pathology laboratories worldwide are using that capability to analyze the SARS-CoV-2 coronavirus and identify variants as they emerge in different parts of the world.
The US Centers for Disease Control and Prevention (CDC) now plans to harness the power of gene sequencing through a new consortium called SPHERES (SARS-CoV-2 Sequencing for Public Health Emergency Response, Epidemiology, and Surveillance) to “coordinate SARS-CoV-2 sequencing across the United States,” states a CDC news release. The consortium is led by the CDC’s Advanced Molecular Detection (AMD) program and “aims to generate information about the virus that will strengthen COVID-19 mitigation strategies.”
The consortium is comprised of 11 federal agencies, 20 academic institutions, state public health laboratories in 21 states, nine non-profit research organizations, and 14 lab and IVD companies, including:
Abbott Diagnostics
bioMérieux
Color Genomics
Ginkgo Bioworks
IDbyDNA
Illumina
In-Q-Tel
LabCorp
One Codex
Oxford Nanopore Technologies
Pacific Biosciences
Qiagen
Quest Diagnostics
Verily Life Sciences
‘Fundamentally Changing How Public Health Responds’
Gene sequencing and related technologies have “fundamentally changed how public health responds in terms of surveillance and outbreak response,” said Duncan MacCannell, PhD, Chief Science Officer for the CDC’s Office of Advanced Molecular Detection (OAMD), in an April 30 New York Times (NYT) article, which stated that the CDC SPHERES program “will help trace patterns of transmission, investigate outbreaks, and map how the virus is evolving, which can affect a cure.”
The CDC says that rapid DNA sequencing of SARS-CoV-2 will help monitor significant changes in the virus, support contact tracing efforts, provide information for developers of diagnostics and therapies, and “advance public health research in the areas of transmission dynamics, host response, and evolution of the virus.”
The sequencing laboratories in the consortium have agreed to “release their information into the public domain quickly and in a standard way,” the NYT reported, adding that the project includes standards for what types of information medical laboratories should submit, including, “where and when a sample was taken,” and other critical details.
Even in its early phase, the CDC’s SPHERES project has “made a tangible impact in the number of sequences we’re able to deposit and make publicly available on a daily basis,” said Pavitra Roychoudhury, PhD (above), Acting Instructor and Senior Fellow at the University of Washington, and Research Associate at Fred Hutchinson Cancer Research Center, in an e-mail to the NYT. “What we’re essentially doing is reading these small fragments of viral material and trying to jigsaw puzzle the genome together,” said Roychoudhury in an April 28 New York Times article which covered in detail how experts are tracking the coronavirus since it arrived in the US. (Photo copyright: LinkedIn.)
Sharing Data Between Sequencing Laboratories and Biotech Companies
The CDC announced the SPHERES initiative on April 30, although it launched in early April, the NYT reported.
According to the CDC, SPHERES’ objectives include:
To bring together a network of sequencing laboratories, bioinformatics capacity and subject matter expertise under the umbrella of a massive and coordinated public health sequencing effort.
To identify and prioritize capabilities and resource needs across the network and to align sources of federal, non-governmental, and private sector funding and support with areas of greatest impact and need.
To improve coordination of genomic sequencing between institutions and jurisdictions and to enable more resilience across the network.
To champion concepts of openness, standards-based analysis, and rapid data sharing throughout the United States and worldwide during the COVID-19 pandemic response.
To provide a common forum for US public, private, and academic institutions to share protocols, methods, bioinformatics tools, standards, and best practices.
To establish consistent data and metadata standards, including streamlined repository submission processes, sample prioritization criteria, and a framework for shared, privacy-compliant unique case identifiers.
To align with other national sequencing and bioinformatics networks, and to support global efforts to advance the use of standards and open data in public health.
Implications for Developing a Vaccine
As the virus continues to mutate and evolve, one question is whether a vaccine developed for one variant will work on others. However, several experts told The Washington Post that the SARS-CoV-2 coronavirus is relatively stable compared to viruses that cause seasonal flu (influenza).
“At this point, the mutation rate of the virus would suggest that the vaccine developed for SARS-CoV-2 would be a single vaccine, rather than a new vaccine every year like the flu vaccine,” Peter Thielen, a molecular biologist at the Johns Hopkins University Applied Physics Laboratory, told the Washington Post.
Nor, he said, is one variant likely to cause worse clinical outcomes than others. “So far, we don’t have any evidence linking a specific virus [strain] to any disease severity score. Right now, disease severity is much more likely to be driven by other factors.”
Fast improvements in gene sequencing technology have made it faster, more accurate, and cheaper to sequence. Thus, as the COVID-19 outbreak happened, there were many clinical laboratories around the world with the equipment, the staff, and the expertise to sequence the novel coronavirus and watch it mutate from generation to generation and from region to region around the globe. This capability has never been available in outbreaks prior to the current SARS-CoV-2 outbreak.
Popularity of the pocket-sized gene-sequencing device continues to prove that DNA testing away from clinical laboratories in remote clinics and outlying field laboratories is not just possible, but in some cases preferable
Since then, MinION’s use worldwide “for a number of biological analysis techniques including de novo sequencing, targeted sequencing, metagenomics, epigenetics, and more” has only expanded, according to multiple sources and ONT’s website.
How Does MinION Work as a Gene Sequencer?
The MinION nanopore sequencing device weighs about 100 grams (less than four ounces), is about the size of a standard deck of cards, operates off a laptop USB plug, and can sequence genetic material in a matter of minutes.
To perform the nanopore sequencing, a strand of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) is pushed through small pores in a membrane. An ionic current is then applied to the material and voltage is implemented to measure any disruptions in the current. The resulting measurement represents an electrical signal that is converted to human-readable sequence.
“It’s like the ultimate barcode,” Gordon Sanghera, PhD, Chief Executive Officer at Oxford Nanopore, told BBC News.
Oxford Nanopore Technologies’ diminutive MinION gene-sequencing device has the capacity to directly recognize epigenetic markers that control gene activity and cellular processes involved in the onset and development of disease. Early detection of cancers, testing for birth defects and infectious diseases, and blood screening are possible future clinical laboratory applications for the MinION. Click on this link to watch video on MinION. (Photo copyright: Oxford Nanopore Technologies.)
Why is MinION Important?
One advantage to this technology is that it has the ability to sequence much longer strands of DNA when compared to existing technologies. The MinION can sequence over a million letters or bases, around 2% of a DNA strand or chromosome with 96% or above accuracy. The device can read remarkably long stretches of consecutive DNA letters. Readouts of several thousand letters are common and the record for the MinION is 882,000 consecutive DNA letters, Technology Review noted.
“One of the most important findings of this research was that, even though the human genome reference was completed or thought to have been completed a while ago, it still contains many missing pieces and we were able to close some of those gaps in the sequence by developing a new method for developing these extremely long reads using nanopore sequencing,” Nick Loman, PhD, Professor of Microbial Genomics and Bioinformatics at the School of Biosciences at the University of Birmingham, UK, told Pharmaphorum. Loman worked on research with Oxford Nanopore on nanopore sequencing.
“We’ve gone from a situation where you can only do genome sequencing for a huge amount of money in well-equipped labs to one where we can have genome sequencing literally in your pocket just like a mobile phone,” Loman told BBC News. “That gives us a really exciting opportunity to start having genome sequencing as a routine tool, perhaps something people can do in their own home.”
Using MinION in the Field
According to the Oxford Nanopore website, the MinION:
Is pocket-sized and portable;
Has up to 512 nanopore channels;
Has a simple 10-minute sample preparation time;
Allows real-time analysis for rapid and efficient results; and,
Is adaptable to direct DNA or RNA sequencing.
The MinION Starter Pack is available for purchase on the company’s website with prices starting at $1,000. The kit includes:
Members of the Zebra Project (above), an international group of scientists, used Oxford Nanopore Technologies’ MinION to sequence genomes during epidemics in Latin America. With just a laptop computer for power, MinION can run complex gene-sequencing and achieve superior results than other similar technologies. It is in use worldwide bringing clinical laboratory testing to patients in remote, outlying locations. (Photo copyright: Ricardo Funari.)
“I think it’s really expanding the arsenal of tools we have to peer into cell biology and the root causes of cancer and various diseases,” Dr. Martin Smith, Head of Genomic Technologies at the center, told Australian Financial Review. “It’s really just starting to open the lid off the jar and peer more deeply into the genomics of the cell.”
Dr. Sanghera hopes the gadget could be utilized in the future to identify common infections at home and help consumers avoid unnecessary trips to doctors, clinics, and hospitals, and avert the misuse and overuse of prescription medications. He also feels MinION has applications outside the healthcare industry, such as detecting the presence of harmful microbes in food and water supplies.
As gadgets like MinION become more popular, the potential to move DNA sequencing closer to the patient (and out of the core lab) has implications for clinical laboratories and anatomic pathology groups. However, core labs would still be a preferred source to collect the raw data, store that data, then do the annotation of the DNA sequences and report the findings to the referring physician.
