Another study in the United Kingdom that also used genomic analysis to understand drug-resistant Shigella produced findings that may be useful for microbiologists and medical laboratory scientists
From the onset of an infectious disease outbreak, public health officials, microbiologists, and clinical laboratory managers find it valuable to trace the origin of the spread back to the “index case” or “patient zero”—the first documented patient in the disease epidemic. Given the decreased cost of genomic analysis and improved accuracy of gene sequencing, infectious disease researchers are finding that task easier and faster than ever.
One recent example is a genomic study conducted at University of Washington (UW) in Seattle that enabled researchers to “retrace” the origin and spread of a “multidrug-resistant Shigellosis outbreak” from 2017 to 2022. “The aim of the study was to better understand the community transmission of Shigella and spread of antimicrobial resistance in our population, and to treat these multi-drug resistant infections more effectively,” the UW scientists stated in a new release.
Shigellosis (aka, bacillary dysentery) is a highly contagious disease of the intestines that can lead to hospitalization. Symptoms include fever, stomach cramps, diarrhea, dysentery, and dehydration.
“Additional analysis of the gut pathogen and its transmission patterns helped direct approaches to testing, treatment, and public health responses,” the UW news release states.
Usually prevalent in countries with public health and sanitation limitations, the “opportunistic” Shigella pathogen is now being seen in high-income countries as well, UW reported.
“You can’t really expect an infectious disease to remain confined to a specific at-risk population. [Shigella infections are] very much an emerging threat and something where our public health tools and therapeutic tools have significant limitations,” infectious disease specialist Ferric Fang, MD (above) told CIDRAP News. Fang is a UW professor of Microbiology and Clinical Laboratory Medicine and a corresponding author of the UW study. (Photo copyright: University of Washington.)
Generally, Shigella infects children, travelers, and men who have sex with men (MSM), the CDC noted.
The UW researchers were motivated to study Shigella when they noticed an uptick in drug-resistant shigellosis cases in Seattle’s homeless population in 2020 at the beginning of the COVID-19 pandemic, Center for Infectious Disease Research and Policy News (CIDRAP News) reported.
“Especially during the pandemic, a lot of public facilities were closed that homeless people were used to using,” infectious disease specialist Ferric Fang, MD, told CIDRAP News. Fang is Professor of Microbiology and Laboratory Medicine at University of Washington and corresponding author of the UW study.
The researchers studied 171 cases of Shigella identified from 2017 to 2022 by clinical laboratories at Harborview Medical Center and UW Medical Center in Seattle. According to CIDRAP News, the UW researchers found that:
46% were men who have sex with men (MSM).
51% were people experiencing homelessness (PEH).
Fifty-six patients were admitted to the hospital, with eight to an intensive care unit.
51% of isolates were multi-drug resistant (MDR).
Whole-Genome Sequencing Reveals Origin
The UW scientists characterized the stool samples of Shigella isolates by species identification, phenotypic susceptibility testing, and whole-genome sequencing, according to their Lancet Infectious Diseases paper. The paper also noted that 143 patients received antimicrobial therapy, and 70% of them benefited from the treatment for the Shigella infection.
Whole-genome sequencing revealed that two strains of Shigella (S. flexneri and S. sonnei) appeared first in Seattle’s MSM population before infecting the PEM population.
The genomic analysis found the outbreak of drug-resistant Shigella had international links as well, according to CIDRAP News:
One S. flexneri isolate was associated with a multi-drug resistant (MDR) strain from China, and
S. sonnei isolates resembled a strain characteristic of a current outbreak of MDR Shigella in England.
“The most prevalent lineage in Seattle was probably introduced to Washington State via international travel, with subsequent domestic transmission between at-risk groups,” the researchers wrote.
“Genomic analysis elucidated not only outbreak origin, but directed optimal approaches to testing, treatment, and public health response. Rapid diagnostics combined with detailed knowledge of local epidemiology can enable high rates of appropriate empirical therapy even in multidrug-resistant infection,” they continued.
UK Shigella Study Also Uses Genomics
Another study based in the United Kingdom (UK) used genomic analysis to investigate a Shigella outbreak as well.
Motivated by a UK Health Security Agency report of an increase in drug-resistance to common strains since 2021, the UK researchers studied Shigella cases from September 2015 to June 2022.
According to a paper they published in Lancet Infectious Diseases, the UK researchers “reported an increase in cases of sexually transmitted S. flexneri harboring blaCTX-M-27 (an antibiotic-resistant gene) in England, which is known to confer resistance to third-generation cephalosporins (antibiotics),” the researchers wrote.
Their analysis of plasmids (DNA with genes having antibiotic resistance) revealed a link in two drug-resistant Shigella strains at the same time, CIDRAP News explained.
“Our study reveals a worsening outlook regarding antimicrobial-resistant Shigella strains among MSM and highlights the value of continued integration of genomic analysis into surveillance and research,” the UK-based scientists wrote.
Current challenges associated with Shigella, especially as it evades treatment, may continue to demand attention from microbiologists, clinical laboratory scientists, and infectious disease specialists. Fortunately, use of genomic analysis—due to its ongoing improvements that have lowered cost and improved accuracy—has made it possible for public health researchers to better track the origins of disease outbreak and spread.
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.
At that reduced cost, clinical laboratories in developing countries with no access to WGS could have it as a critical tool in their fight against the spread of deadly bacteria and viruses
New research into a low-cost way to sequence bacterial genomes—for as little as $10—is predicted to give public health authorities in low- and middle-income countries (LMICs) a new tool with which to more quickly identify and control disease outbreaks.
This new approach offers an alternative to more expensive Whole genome sequencing (WGS) methodologies, which clinical laboratories in developed countries typically use to identify and track outbreaks of infectious diseases. And with SARS-CoV-2 variants resulting in increased COVID-19 infections, the ability to perform low-cost, rapid, and accurate WGS is becoming increasingly important.
But for many developing countries that need it the most, the cost of WGS has kept this critical technology out of reach.
Now, a global consortium of scientists has successfully established an efficient and inexpensive pipeline for the worldwide collection and sequencing of bacterial genomes. The large-scale sequencing method could potentially provide researchers in LIMCs with tools to sequence large numbers of bacterial and viral pathogens. This discovery also could strengthen research collaborations and help tackle future pandemics.
The team of scientists, led by researchers at the Earlham Institute and the University of Liverpool, both located in the UK, are confident their technology can be made accessible to clinical laboratories in LMICs around the globe.
“It has been 26 years since the first bacterial genome was sequenced, and it is now possible to sequence bacterial isolates at scale,” Neil Hall, PhD (above), director of the Earlham Institute and one of the authors of the study, told Genetic Engineering and Biotechnology News. “However, access to this game-changing technology for scientists in low- and middle-income countries has remained restricted. The need to ‘democratize’ the field of pathogen genomic analysis prompted us to develop a new strategy to sequence thousands of bacterial isolates with collaborators based in many economically challenged countries.” (Photo copyright: Earlham Institute.)
Streamlining Collection and Sequencing
The international team of scientists aimed their innovative WGS approach at streamlining the collection and sequencing of bacterial isolates (variants). The researchers collected more than 10,400 clinical and environmental bacterial isolates from several LMICs in less than a year. They optimized their sample logistics pipeline by transporting the bacterial isolates as thermolysates from other countries to the UK. Those isolates were sequenced using a low cost, low input automated method for rapid WGS. They then performed the gene library construction and DNA sequencing analysis for a total reagent cost of less than $10 per genome.
The scientists focused their research on Salmonella enterica, a pathogen that causes infections and deadly diseases in human populations. Non-typhoidal Salmonella (NTS) have been associated with enterocolitis, a zoonotic disease in humans linked to industrial food production.
Because the disease is common in humans, there have been more genome sequences generated for Salmonella than any other type of germ.
“In recent years, new lineages of NTS serovars Typhimurium and Enteritidis have been recognized as common causes of invasive bloodstream infections (iNTS disease), responsible for about 77,000 deaths per year worldwide,” the researchers wrote in their Gen Biology paper. “Approximately 80% of deaths due to iNTS disease occurs in sub-Saharan Africa, where iNTS disease has become endemic.”
Increasing Access to Genomics Technologies in Developing Countries
The research consortium 10,000 Salmonella Genomes Project (10KSG) led the large-scale WGS initiative. The alliance involves contributors from 25 institutions in 16 countries and was designed to generate information relevant to the epidemiology, drug resistance, and virulence factors of Salmonella using WGS techniques.
“One of the most significant challenges facing public health researchers in LMICs is access to state-of-the-art technology, Jay Hinton, PhD, Professor of Microbial Pathogenesis at the University of Liverpool and one of the paper’s authors, told Technology Networks. “For a combination of logistical and economic reasons, the regions associated with the greatest burden of severe bacterial disease have not benefited from widespread availability of WGS. The 10,000 Salmonella genomes project was designed to begin to address this inequality.”
The authors noted in their study that the costs associated with sequencing have remained high mostly due to sample transportation and library construction and the fact that there are only a few centers in the world that have the ability to handle large-scale bacterial genome projects.
“Limited funding resources led us to design a genomic approach that ensured accurate sample tracking and captured comprehensive metadata for individual bacterial isolates, while keeping costs to a minimum for the Consortium,” Hall told Genetic Engineering and Biotechnology News(GEN). “The pipeline streamlined the large-scale collection and sequencing of samples from LMICs.”
“The number of publicly available sequenced Salmonella genomes reached 350,000 in 2021 and are available from several online repositories,” he added. “However, limited genome-based surveillance of Salmonella infections has been done in LMICs, and the existing dataset did not accurately represent the Salmonella pathogens that are currently causing disease across the world.”
The $10 cost is designed to help healthcare systems in developing countries identify the specific genetic composition of infectious diseases. That’s the necessary first step for developing a diagnostic test that enables physicians to make an accurate diagnosis and initiate appropriate therapy.
“The adoption of large-scale genome sequencing and analysis of bacterial pathogens will be an enormous asset to public health and surveillance in LMI countries,” molecular microbiologist Blanca Perez Sepulveda, PhD, told GEN. Sepulveda is a postdoctoral Researcher at the University of Liverpool and one of the authors of the study.
Improvement in next-generation sequencing technology has reduced costs, shortened turnaround time (TAT), and improved accuracy of whole genome sequencing. Once this low-cost method for collecting and transporting bacterial sequences becomes widely available, clinical laboratories in developing countries may be able to adopt it for genome analysis of different strains and variants of bacteria and viruses.
Scientists working to sequence all 1.66 million animal species say this is a missed opportunity to better understand our own genetics; such research would identify biomarkers useful for clinical laboratory testing
For 23 years, the world’s genomic scientists have been on a mission to sequence the genomes of all animal species. And they’ve made great progress. However, according to a recent study conducted by researchers at Washington State University (WSU) and Brigham Young University (BYU), only a fraction of the sequences are from invertebrate species. And that, according to the study’s authors, is “overlooking huge swathes of diversity and opportunity.”
The push to sequence the whole genomes of all animals began in 1998 with the sequencing of the Caenorhabditis elegans roundworm, according to a WSU news release. It was the first animal genome sequence, but it was not to be the last. Nearly 25 years later, genomic scientists have sequenced about 3,300 animal genomes. And while that’s a lot of genomic sequences, it’s a drop in bucket of the approximately 1.7 million animal species on the planet.
But here’s where the missed opportunity comes in. According to the WSU news release, “Vertebrates account for 54% of all genome sequencing assemblies, despite representing only 3.9% of animal species. In contrast, the invertebrates of the Arthropoda phylum, which includes insects and spiders, comprise only 34% of current datasets while representing 78.5% of all species.”
“We are interested in ourselves, and that’s not necessarily a bad thing,” said Paul Frandsen, PhD (above), in the news release. Frandsen is Assistant Professor of Genetics, Genomics, and Biotechnology at Brigham Young University and one of the study authors. “But to begin to understand entire ecosystems,” he continued, “we have to start sampling more of the variety of life to gain a clearer picture. Vertebrates are important components of ecosystems, but arguably insects and many other small creatures probably play an even more important role because they’re down at the base of the food web.” (Photo copyright: Brigham Young University.)
The scientists analyzed the best available genome assemblies found in GenBank, the world’s most extensive genetic database. They found that 3,278 unique animal species across 24 phyla, 64 classes, and 258 orders have been sequenced and assembled to date.
They also found that sequencing efforts have focused heavily on species that most resemble humans. The Hominidae, a taxonomic family of primates that includes humans as well as great apes, bonobos, chimpanzees, orangutans, and gorillas, has the most contiguous genome data assembled.
The team discovered that vertebrates account for 54% of the animal genome sequencing that has been performed even though they make up less than four percent of known animal species. By comparison, invertebrates of the Arthropoda phylum, which represent 78.5% of all animal species, comprise only 34% of the completed animal genome sequencing. And yet, the Arthropoda phylum is the largest phylum in the animal kingdom and includes insects, spiders, scorpions, centipedes, millipedes, crabs, crayfish, lobsters, and barnacles.
“With genome assemblies accumulating rapidly, we want to think about where we are putting our efforts. It’s not being spread evenly across the animal tree of life,” said lead author Scott Hotaling, PhD, post-doctoral researcher at WSU, in the news release. “Invertebrates are still very underrepresented, which makes sense given that people seem to care more about vertebrates, the so-called ‘charismatic megafauna.’”
The team discovered that only five arthropod groups: ants, bees, butterflies, fruit flies, and mosquitos, were well represented in genome sequencing. The longest genome sequenced so far belongs to the Australian lungfish, the only surviving member of the family Neoceratodontidae.
1,100 Years to Sequence All Eukaryotic Life
The scientists also discerned that animal genome assemblies have been produced by 52 countries on every continent with permanent inhabitants. The majority of animal genome sequencing (77%) that is being performed is mostly occurring in developed countries located in the Northern Hemisphere, often referred to as the Global North. Nearly 70% of all animal genome assemblies have been produced by just three countries: the United States, China, and Switzerland.
There are geographic differences between regions regarding the types of animals being sequenced and assembled with North America concentrating on mammals and insects, Europe focusing on fish, and birds being the main type of animals sequenced in Asia.
The scientists would like to see more animal genome sequencing happening in countries from the Global South, or Southern Hemisphere, particularly in tropical regions that contain a myriad of diversity among animal species.
“If we want to build a global discipline, we need to include a global people,” Hotaling said. “It’s just basic equity, and from a pure scientific standpoint, the people who live in areas where species are being sequenced have a lot of knowledge about those species and ecosystems. They have a lot to contribute.”
But the WSU/BYU scientists found that many species in GenBank only have low-quality assemblies available. They noted that “the quality of a genome assembly is likely the most important factor dictating its long-term value.”
Fortunately, several animal genome sequencing ventures have been announced in recent years, so the amount of available data is expected to rise exponentially. These projects include:
The Earth BioGenome Project (EBP) which aspires to sequence and catalog the genes of all the eukaryotic species on the planet within ten years.
The Vertebrate Genomes Project which seeks to generate high-quality assemblies for 70,000 extant vertebrate species.
The Bird 10K Project that seeks to generate assemblies for all extant birds.
The i5K Project which plans to produce 5,000 arthropod genome assemblies.
The authors of the PNAS paper noted that there are currently only about four genome assemblies happening each day and, at that rate, the sequencing of all eukaryotic life will not be completed until the year 3130.
So, microbiologists, clinical laboratory professionals, and genomic scientists have plenty of time to get up to speed.
GISAID hosts a vast, open database of genomic sequences of SARS-CoV-2 coronavirus samples, and medical laboratory scientists in countries across the globe are contributing
Clinical laboratories around the world have been contributing to the global scientific community’s database of knowledge about SARS-CoV-2, the coronavirus that caused the COVID-19 pandemic, and its variants, through an ingenious and crucial network known as GISAID. This cooperative sharing of the coronavirus’ genetic data (now four million genomic sequences strong) has greatly contributed to understanding the spread of infections and progress obtained in developing effective treatments and vaccines.
Headquartered in Munich, Germany, GISAID, which stands for Global Initiative on Sharing Avian Influenza Data, was created in 2008 during the Avian Influenza (Bird Flu) pandemic. The GISAID initiative promotes “the rapid sharing of data from all influenza viruses and the coronavirus causing COVID-19. This includes genetic sequence and related clinical and epidemiological data associated with human viruses, and geographical as well as species-specific data associated with avian and other animal viruses, to help researchers understand how viruses evolve and spread during epidemics and pandemics,” according to the GISAID website.
Clinical pathologists are likely familiar with GISAID. The initiative has become an indispensable tool for researchers battling SARS-CoV-2. GISAID allows scientists and organizations worldwide to upload genetic sequences of COVID-19 samples. Those sequences can then be used in research for treatments, vaccines, and to track emerging variants. The information is invaluable, freely available, and represents the collaborative efforts of scientists around the world in the fight against COVID-19 and other infectious diseases.
An article published in The World, titled, “From Congo to Chile, Small Labs Are Playing a Growing Role in Global Understanding of COVID,” noted that more than four million genomic sequences have been submitted as of October 15, 2021. The more countries around the world that submit sequences to GISAID, the more understanding scientists have of how the virus is mutating. And, as the cost of performing genomic sequencing declines, the number of countries submitting genomes of SARS-CoV-2 to GISAID is rising.
How GISAID Ensures Contributors Receive Credit for Their Work
One of the reasons that GISAID has been so successful in gathering data is that it requires anyone who uses data downloaded from the massive database to give credit to the person or organization who uploaded it. In other words, if a scientist in the United Kingdom (UK) does breakthrough research using genomes that were originally uploaded to GISAID by a scientist in the Congo, the UK scientist must credit the work of the scientist from the Congo.
Other genomic databases do not have this requirement and genetic researchers are often hesitant to share information due to fear their work will be co-opted by others. According to The World, scientists in lower income countries are particularly vulnerable to having their work appropriated.
Even worse is having one’s work appropriated, used to create a product, and then not being given access to that product.
“Unfortunately, we’ve seen also the situation whereby people have leveraged that data and created the solution and refused to share the solution with those that shared the data,” virologist Christian Happi, PhD (above), who directs the African Center of Excellence for Genomics of Infectious Diseases (ACEGID) at Redeemer’s University in Nigeria, told The World. “And that is definitely going to roll back this whole open data sharing and access principle.” Happi is also a Visiting Scientist in the Department of Immunology and Infectious Diseases at Harvard’s T.H. Chan School of Public Health. (Photo copyright: Pius Utomi Ekpei/AFP/News 24.)
That is why GISAID’s policy of giving credit is so important, as molecular biologist Francine Ntoumi, PhD, told The World. “This means that we are going to participate in the game. We are able to say what is circulating. You are no more an observer and I think it makes a difference.” Ntoumi is Founder and Executive Director of the Congolese Foundation for Medical Research (CFMR) in the Republic of Congo, a lecturer in Immunology at Marien Ngouabi University, and Associate Professor and Head of a Research Group at the Institute of Tropical Medicine at the University of Tübingen, Germany.
The guarantee that credit will be given softens some of those fears and explains why the GISAID database is so vast, and increasingly contains sequences from scientists in Africa, South American, and other places where genomic sequencing was not widespread prior to the pandemic. Information from all over the world is crucial for scientists monitoring the mutations of the SARS-CoV-2 coronavirus.
Criticisms of GISAID
The fact that more countries are contributing to the GISAID database is certainly a positive, but the non-profit is not without its critics. There have been complaints about the lack of transparency, and some researchers claim to have had their access denied to the data without any explanation.
An article published in Science reported that “Scientists live in fear of losing access to the GISAID database.”
One scientist who requested anonymity told Science, “I am so tired of being scared all the time, of being terrified that if I take a step wrong, I will lose access to the data that I base my research on. [GISAID] has that sword hanging over any scientist that works on SARS-CoV-2.”
In response to these criticisms, GISAID said in a statement, “Any individual who registers with GISAID and agrees to the GISAID terms of use will be granted access credentials. … On rare occasions, GISAID has found it necessary to temporarily suspend access credentials to protect the GISAID sharing mechanism,” The World reported.
The strict sharing rules may be necessary to encourage researchers in lower income countries to contribute their genomic data on SARS-CoV-2. Charles Rotimi, PhD, a geneticist at the National Human Genome Research Institute (NHGRI), told Science, “To make scientists, especially from developing countries, more comfortable—making sure that they are recognized in the work that they are doing—sometimes you have to create an extra layer [of protection].”
GISAID has certainly accomplished much in its assembling four million SARS-CoV-2 genetic sequences. The initiative’s efforts have contributed to a substantial increase in the number of countries around the world that now have gene sequencing capabilities.
This is another illustration for clinical laboratory managers and pathologists of how continual technology advances in gene sequencing equipment and data analysis software make it significantly cheaper, faster, and more accurate to do genetic sequencing. This was not true, just a few years ago.
