With the decreasing cost of nucleic acid sequencing and the rapid development of technology, macro-genome sequencing is expected to enter clinical applications. In addition to studying the human microbiome, the application of macrogenome sequencing is changing the way doctors diagnose and treat diseases. So what are the current problems facing macrogenome sequencing? And how should it be developed in the future?
Today, we jointly focus on the application of macrogenome sequencing in clinical settings, and hope this article can bring some inspiration and help to industry stakeholders and readers.
Macrogenomics Detection Technology
As nucleic acid sequencing has become faster, more accurate, and cheaper, whole genome sequencing has gone from an exciting technological breakthrough to a mundane affair. Macrogenome sequencing now allows researchers to study the entire collection of genomes in a microbiome at the same time. This is highly applicable for understanding the human or animal microbiome, diagnosing infections, or monitoring emerging pathogens.
In 2014, the first case report confirmed that macrogenomics can be successfully applied to the treatment of critically ill patients: Dr. Charles Y. Chiu, Professor of Laboratory Medicine and Medicine/Infectious Diseases at the University of California, San Francisco (UCSF), led his scientific team to use sequencing technology to detect Leptospira in cerebrospinal fluid samples Leptospira was detected, while traditional infectious disease testing did not reveal any problems. The patient was subsequently treated with appropriate antibiotics and recovered.
Since then, a variety of macrogenomic services have emerged: UCSF's Next Generation Precision Diagnostics Center offers macrogenomic sequencing of cerebrospinal fluid or plasma samples in 1-2 week cycles; Karius offers a test called Karius, which sequences free cellular DNA in plasma to detect over 1,000 pathogens, with results typically returned in a day or two; Arc Bio offers a fully integrated workflow, Galileo ONE, which is compatible with Illumina sequencers, and Arc Bio claims that Galileo ONE detects over 1,000 pathogens through a fully integrated workflow. Arc Bio offers a fully integrated workflow, Galileo ONE, which is compatible with Illumina sequencers and does not require bioinformatics expertise, and Arc Bio claims that Galileo ONE enables the analysis of DNA and RNA, or both, from a single sample by analyzing by analyzing DNA and RNA, or both, to detect and quantify more than 99% of known human pathogens from a single sample.
Laboratories and hospitals around the world are actively testing clinical macrogenomics with promising results.
Dr. Etienne Ruppé, professor of medicine at the University of Paris, said, "This is still an emerging field, but it is now widely considered to be the emerging frontier in diagnosing infectious diseases." However, he also hints, "But there are still many obstacles to its general application in the laboratory."
The gold standard for diagnostic microbiology is the analysis of microorganisms cultured from patient samples. A quick alternative is the use of PCR assays, which can identify DNA or RNA of microorganisms; however, rapid molecular detection techniques are limited to the detection of pathogens on the candidate list.
In contrast, macrogenome sequencing can characterize all DNA or RNA in a sample. Although sequencing is not as fast as PCR assays, it is much faster than culturing and analyzing cultures.
When the COVID-19 outbreak hit, the opportunity for macrogenomics to diagnose the risk of nosocomial infections in vulnerable populations presented itself. Dr. Jonathan Edgeworth, a pathologist and director of the Centre for Clinical Infection and Diagnostic Research (CIDR) at King's College London, observed an increased risk of hospital-acquired infections when COVID-19 patients congregate in ICUs.
Intubation and other interventions also put patients at risk for infection. Pathogens can be transmitted from patient to patient through devices that are not fully sterilized. At times, the risk of infection is also increased when an otherwise harmless microorganism is introduced into a new environment, such as the lungs or blood.
Often, clinicians will administer antibiotics to patients based on the most likely guess, rather than waiting for the analysis results to come back. However, misuse of antibiotics may lead to the development of drug-resistant pathogens.
There is a lack of comprehensive microbiological information to guide you in deciding what clinical treatment strategy to use," Edgeworth said. The advent of nanopore sequencing technology makes us think it has the potential to provide the information that culture means provide."
Nanopore sequencing allows for real-time analysis of long DNA fragments, and long sequences allow for better species identification. "This is very useful for us," emphasizes Dr. Themoula Charalamous, a postdoctoral researcher at CIDR, "and we want our workflow to be as fast as possible. We want to get results the same day."
Although multiplex PCR assays for 20 or more species are usually sufficient, they still create a degree of uncertainty if the infection is not caused by one of the microbes on the candidate list. edgeworth insists, "When you're in the intensive care unit, that's not enough. The thing about macrogenomics is that it sequences all the genomes. It's the equivalent of a culture that would normally take three days to get, given to you on the same day."
A study involving Charalampous and Edgeworth has shown that nanopore sequencing can detect pathogens in real clinical ICU settings with high sensitivity and predict drug resistance. Not only does the nanopore workflow provide same-day results, but EPI2ME, an automated data analysis platform provided by Oxford Nanopore Technologies, also eliminates the need for bioinformatics experts to interpret the results.
Still, it's a bit too labor-intensive for broad clinical applications, Charalampous notes: "It takes about eight hours, most of which is manual time. It requires good molecular biology skills."
Obstacle 2: Beware of "kitome"
In a study using nanopore sequencing, researchers intentionally selected patients flagged by clinicians as likely to be infected. edgeworth says, "We didn't want too many negative results. This was a proof-of-concept study."
However, in routine macrogenomic analyses, it is often necessary to include negative control samples to identify microorganisms present in the DNA extraction kit, known as the kitome. "When we sequence samples that are actually negative, macrogenomic sequencing will also have a bacterial signal," Ruppé says. "The DNA is always there. That's what we call the kitome."
To complicate matters, the kitome can produce different bacteria each time. Treating the kitome cannot be as simple as ignoring some known contaminants. "It's a very big challenge for us," Ruppé insists, "that the bacteria found in the kitome could also be pathogens."
For example, Propionibacterium acnes is usually found on the skin, so they are usually likely to be found in kitome. however, "it is also a notorious pathogen in bone and joint infections of the shoulder." Ruppé writes. If a potentially infected shoulder sample is positive for Propionibacterium acnes, it can be difficult to distinguish a true positive from a contaminant.
This is a major issue in macrogenomics. In most cases, traditional microbiology techniques still offer advantages in terms of cost, extensive clinical validation, broad applicability, and accurate diagnosis of most typical infections. Where macrogenomics really shines, at least for now, is in solving certain challenging cases where traditional approaches have failed to find answers.
"When all else fails, clinical macrogenomics has been positioned as the 'diagnostic tool of last resort with potential,'" Ruppé argues, "but if everything else fails, then there's a good chance the sample is supposed to be negative, so you have to take contaminants seriously."
As macrogenomic technology becomes more automated and inexpensive, its widespread use becomes more feasible. For example, macrogenomics can be used to advantage in early patient care. "That's one of the problems with clinical macrogenomics right now," Ruppé says, "should we only use it as a last resort test? And what are the benefits of using it in the early stages?"
There is growing evidence that clinical macrogenomics should be considered along with existing approaches, not just after all other approaches have failed. Dr. Natacha Couto, associate researcher at the University of Bath, said there are many reports in the literature that suggest that macrogenomics is more sensitive than culture, and many that suggest otherwise. However, Couto emphasized that many studies have shown that macrogenomics does complement the information provided by culture means.
Couto notes, "There are many examples ...... of macrogenomics discovering potential microbes that would not have been discovered by culture alone. Just like when PCR was introduced, it became a complement to culture means, this will happen in macrogenomics as well."
Natacha Couto of the University of Bath predicts that macrogenomics may eventually become a complementary tool to traditional microbial culture. Next-generation sequencing (NGS) methods are faster than culture methods, and NGS, unlike PCR, does not need to be limited to a pre-selected set of targets. Macrogenomic sequencing can rapidly identify pathogens present in a sample and detect the presence of antimicrobial resistance genes, providing actionable information for patient treatment.
Earlier use of macrogenomics can prevent infections before they become symptomatic, save money, shorten hospital stays, and reduce patient stress. Dr. David Haslam, a pediatrician and director of the Microbial Genomics and Macrogenomics Laboratory at Cincinnati Children's Hospital Medical Center, sees the potential for the use of macrogenomics in monitoring susceptible patients after antibiotic therapy.
"The microbiome may somehow become abnormal, and we're trying to use that as a predictor of future infections." Haslam said.
He gave the example of a patient whose normal, diverse microbiome was disrupted after a course of antibiotics and a particular strain of E. coli began to dominate. When the patient developed a severe bloodstream infection, sequencing showed that the invading bacteria were the same strain as the bacteria that had taken over the gut microbiome.
Thus, monitoring the gut microbiota can provide early warning of such bacterial infections, Haslam added, adding that the Karius test can be used to detect bacterial DNA in the blood before any external infection occurs.
Haslam explains, "[Monitoring the microbiome] is very exciting because it may help you intervene before a patient gets sick."
Another issue, Couto noted, is that macrogenomics can extract nucleic acid signatures from already dead organisms. Consider a case where a patient becomes infected and begins treatment, but the symptoms continue. Even if the treatment is effective, she says, macrogenomic sequencing can extract residual DNA from the dead organism, Couto insists, depending on "the clinician should combine all available data, including relevant symptom data and the laboratory methods that have been used, to determine whether an infection has actually occurred."
Barrier 4: Differentiating between similar genes and bacteria
Macrogenomics provides us with a better way to detect antibiotic resistance. "PCR assays provide fast and accurate information about antibiotic resistance genes, but these assays focus on only a few antibiotic resistance genes," Haslam explained, "and in theory, macrogenomics can study every antibiotic resistance gene. "
However, he noted that this needs to be expanded with great caution; PCR assays can focus on genes that are strongly associated with antibiotic resistance, while macrogenomics can capture less defined genes. For example, macrogenomics needs to distinguish between members of gene families that share some sequence similarity but have different levels of resistance.
"It's mostly a bioinformatics challenge and a computational challenge," Haslam said. "I think it's promising, though it may take a few more years, but I think machine learning will be the answer to these gray areas.
Another such gray area is distinguishing between similar bacterial species - small genomic changes can mean the difference between harmless commensal pathogens and dangerous ones. It is difficult to separate closely related species using macrogenomics techniques because all genomes are mixed together in the sample.
According to a recent study, researchers from the U.S. Department of Agriculture and the University of California, San Diego, overcame this problem by using PacBio's HiFi technology, a sequencing technique that produces long read-length sequences with greater than 99.9 percent accuracy. When the researchers used this technology to sequence the macrogenome of sheep feces, they were able to assemble a truly complete bacterial genome without being trapped by highly repetitive segments. They used high-throughput chromosome conformation capture (Hi-C) technology to classify overlapping groups (contigs) and then used an algorithm from MAGPhase to resolve closely related haplotypes.
Ultimately, the study identified 428 genomes with more than 90% completeness, 44 of which were single loop contigs. these findings suggest that a complete macrogenomic process can successfully evaluate many macrogenomes, including the human macrogenome.
One of the study's leaders, Dr. Pavel A. Pevzner, a professor of computer science at the University of California, San Diego, says macrogenomic technology can help us deal with situations like the 2011 E. coli outbreak in Germany.
Sequencing the pathogenic E. coli that caused the outbreak wasn't so easy," he recalls. I am sure that if there is a future bacterial outbreak, people will use this macrogenomic approach to define the disease-causing species. This cost is insignificant compared to the billions of dollars in potential losses from an outbreak."
Pevzner declares, "Macrogenomics is entering a new era. Our research is just a signal that this new era has arrived."
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