Metagenomic Next Generation Sequencing: How Does It Work and Is It Coming to Your Clinical Microbiology Lab? | ASM.org (2022)

Next generation sequencing (NGS) methods started to appear in the literature in the mid-2000s and had a transformative effect on our understanding of microbial genomics and infectious diseases. There is nonetheless considerable controversy on how, when, and where next generation sequencing will play a role in the clinical diagnostic laboratory. A deep dive point-counterpoint discussion from the Journal of Clinical Microbiology discusses the challenges and opportunities that may come with introduction of metagenomic next generation sequencing (mNGS) into routine laboratories. What exactly is mNGS and how is it different from the many other nucleic acid technologies out there?

What is Metagenomic Next Generation Sequencing?

Next generation sequencing is any of several high-throughput sequencing methods whereby billions of nucleic acid fragments can be simultaneously and independently sequenced. Contrast this technique to classical methods such as Sanger sequencing (also known as dideoxynucleotide chain termination sequencing), which processes one nucleotide sequence per reaction.

(Video) Metagenomics principles and workflow

To characterize a bacterial genome using NGS, for example, the genome is split into multiple fragments that produce sequences or reads ranging from hundreds to tens of thousands of bases in length. The sequences are assembled into a single genome using computational approaches. Several overlapping sequence reads are pieced together to produce a single longer sequence called a contig. There are often gaps between contigs and although high-fidelity longer sequence reads would be the ideal method of sequencing, platforms that produce shorter reads are generally less costly and the overlap in sequences makes them more accurate. The constructed genome (likely containing gaps) is aligned to a reference database for identification of the organism. This technology represents a substantial advance over the early days of sequencing when a single bacterial genome project could take several years.

Metagenomic NGS (mNGS) is simply running all nucleic acids in a sample, which may contain mixed populations of microorganisms, and assigning these to their reference genomes to understand which microbes are present and in what proportions. The ability to sequence and identify nucleic acids from multiple different taxa for metagenomic analysis makes this a powerful new platform that can simultaneously identify genetic material from entirely different kingdoms of organisms.
The possible clinical applications are tremendous, including diagnosis of infectious diseases, outbreak tracking, infection control surveillance, and mutation and pathogen discovery, among many others. mNGS, sometimes called shotgun sequencing, of clinical samples has been applied to various sample types including cerebrospinal fluid, blood, respiratory samples, gastrointestinal fluid, and ocular fluid.

(Video) Next Generation Sequencing | Metagenomics Sequencing

Metagenomic Next Generation Sequencing: How Does It Work and Is It Coming to Your Clinical Microbiology Lab? | ASM.org (1)

What are the Benefits of Metagenomic Next Generation Sequencing?

The largest strength of mNGS is that it is an unbiased hypothesis-free diagnostic method, unlike targeted polymerase chain reaction (PCR) methods that rely on primers for identification of specific targets to be amplified and detected. Even universal or broad-range PCR methods are not sufficiently broad to be considered metagenomic, as they use specific primers of conserved 16S ribosomal RNA (rRNA) gene and internal transcribed spacer (ITS) sequences to amplify distinctive nucleic acid sequences that can be bioinformatically classified into bacteria/archaea, or fungi respectively.

Universal primers also pose a problem when diagnosing polymicrobial infections with molecular tests. If polymicrobial populations are present when using 16S sequencing, multiple base-calls will be made per nucleotide, producing a mixed nucleotide chromatogram that cannot be interpreted. While there are de-convolutional computational methods available to predict organisms identified, these are not in standard use for many laboratories, which often reflex to next-generation sequencing of the 16S gene for polymicrobial samples.

(Video) The Expanding Role of Diagnostic Next Generation Sequencing NGS in the Clinical Microbiology...

What are the Challenges of Metagenomic Next Generation Sequencing?

Despite the potential of mNGS, there are many barriers to clear before the technology can become part of the mainstream laboratory, as well as gaps in our understanding about its diagnostic utility. Major reservations include the interpretation of findings (distinguishing contamination and colonization from true pathogens), selection and validation of databases used for analyses, and prediction (or lack thereof) of antimicrobial susceptibilities. A common perception is that mNGS is so incredibly sensitive that it will reveal a diagnosis when all other testing is negative. While mNGS may be analytically more sensitive than standard culturing methods in some cases, the necessary removal of vast amounts of human nucleic acid during sequencing preparation and (by computational methods) during the post-analytic process, can decrease the sensitivity in comparison to targeted PCR approaches for many organisms.

The specificity of mNGS remains the proverbial elephant in the room. Contamination of samples during specimen collection is a large concern given the increased analytical sensitivity of mNGS in comparison to standard culture methods, and there needs to be a validated quality-control process in place for steps from assessing reagent purity to measuring adequate genome coverage controls. Furthermore, with some Illumina platforms, the wrong barcode indices can be designated, leading to false positives on sequencing data. Bioinformatic quality controls are needed to ensure that high quality and validated genomes are available with minimal database errors and there would ideally be bioinformatic personnel available to interpret sequencing results for each test, which is not available at most clinical microbiological laboratories. The Federal Drug Administration (FDA) has collaborated with other federal agencies to curate a database entitled FDA-ARGOS (FDA-database for regulatory-grade microbial sequences), which has been useful to ensure that current mNGS results are reliable and accurate, but these resources need to be updated and maintained.

(Video) Applications of Clinical Microbial Next-Generation Sequencing - American Academy of Microbiology

The greater question remains surrounding the clinical specificity of mNGS: Are the detected sequences from pathogens that are contributing to the patient’s current disease? The analytical specificity of the mNGS testing can be addressed with rigorous controls throughout specimen collection, sequencing library preparation, assay run, and bioinformatic classification, but clinical specificity is not directly addressed by these approaches. Questions that can help determine clinical utility and applicability include: How can we distinguish organisms related to transient bacteremia from oral/gastrointestinal flora or skin colonizers in blood/plasma mNGS testing? How should sequencing depth be reported and how reliable is the relationship of sequence depth to true infection? Does this relationship differ by pathogen/host? How long is the expected detectable half-life of a pathogen by mNGS once the patient is receiving appropriate curative therapy? Studies on clinical utility and cost-effectiveness are greatly needed despite the indisputable power of this technology from a research and discovery perspective.

It’s also worth pointing out that there are no currently FDA-cleared or approved mNGS tests that can be sent for microbial testing, although there are laboratories certified under the Clinical Laboratory Improvement Amendments of 1988 (CLIA ’88) which offer testing on clinical samples. To date, only a few diagnostic NGS systems have been cleared by the FDA for oncological testing or detection of cystic fibrosis, for example. A recent review describes in detail many of the regulatory hurdles and considerations that will need to be addressed before mNGS could enter mainstream clinical diagnostic laboratories as an FDA-validated test.

(Video) WEBINAR: Cost-effective, Miniaturized Next-Generation Sequencing for Microbiome and Metagenomics

In summary, while mNGS testing may likely play a major role in the microbiological diagnostic workflow in the future, particularly as sequencing and bioinformatic processing power evolves, this remains a high-complexity technology for which the clinical utility in our current medical practice environment remains uncertain. Although mNGS testing may offer novel and exciting diagnostic clinical opportunities in the near future, none of it will likely replace an astute clinician anytime soon.


The above represent the views of the author and does not necessarily reflect the opinion of the American Society for Microbiology.

(Video) Using Next Generation Sequencing to Identify Pathogens

FAQs

How does metagenomic sequencing work? ›

In metagenomics, the genetic materials (DNA, C) are extracted directly from samples taken from the environment (e.g. soil, sea water, human gut, A) after filtering (B), and are sequenced (E) after multiplication by cloning (D) in an approach called shotgun sequencing.

Why is metagenomics important in microbiology? ›

Metagenomics allows us to discover new genes and proteins or even the complete genomes of non-cultivable organisms in less time and with better accuracy than classical microbiology or molecular methods.

What is the purpose of metagenomic analysis? ›

Metagenomics is the study of the structure and function of entire nucleotide sequences isolated and analyzed from all the organisms (typically microbes) in a bulk sample. Metagenomics is often used to study a specific community of microorganisms, such as those residing on human skin, in the soil or in a water sample.

What is metagenomic sequence? ›

Sequence-Based Metagenomic Analysis. Sequence-based metagenomics is used to collect genomic information from microbes without culturing them. In contrast to functional screening, this approach relies on sequence analysis to provide the basis for predictions about function.

What are the 3 steps to metagenomics study? ›

Basically, metagenomic studies include three important steps, including the extraction of desired DNA from an environmental sample, the construction of metagenomic libraries via transformation techniques, and the screening of desired clones, genes, or products and the identification of the desired product. ...

What is next generation sequencing metagenomics? ›

What is Metagenomic Next Generation Sequencing? Next generation sequencing is any of several high-throughput sequencing methods whereby billions of nucleic acid fragments can be simultaneously and independently sequenced.

What is the biggest advantage of metagenomics? ›

Like other molecular biological tools (MBTs), metagenomics eliminates the need to grow organisms in the laboratory, thus eliminating the biases associated with traditional, cultivation-based methods like plate counts.

What is metagenomics and its applications? ›

Metagenomics is the study of a collection of genetic material (genomes) from a mixed community of organisms. In short, metagenomics is a new way to study microorganisms in a specific environment by using functional gene screening or sequencing analysis.

What is metagenomics microbiome? ›

Metagenomics: towards a better understanding of the human gut microbiome. Metagenomics was first described in 1998 by Handelsman and Rodon. 35 ,36 It was defined as analysis of the collective genomes that are present in a defined environment or ecosystem, hence giving insight into functions of non-cultivated bacteria.

What are the principle of metagenomics? ›

Metagenomics, the principle of which relies on the genomic analysis of a sample from a complex environment containing more than one microorganism, provides a view of the composition of this sample. Metagenomic studies became increasingly accessible with the advent of Next Generation Sequencing (NGS) [4].

Why is metagenomics so important for exploring microbial diversity? ›

Metagenomics relies on isolating the genetic material from an environmental sample to determine the amount and variety of microbes present without needing to culture anything at all.

What technique is used for metagenomics? ›

There are two common methods used in metagenomics: shotgun sequencing and directed sequencing. In shotgun sequencing, scientists sequence many small sections of the genome and reconstruct the entire genome by figuring out how these small sections fit together.

What are the applications of metagenomics in health? ›

It can be used to determine gut microbial species and their abundance, and allows to monitor human health and well-being. Metagenomics sheds the light into the development of probiotics. Monitoring of human-associated bacterial communities allows to establish ways to modulate them, so as to optimize human health.

What is the difference between microbiome and metagenomics? ›

The microbiome definition in biology refers to the microorganisms and their genes whereas the microbiota only refers to the microbes themselves. If you just want to talk about all the genes in an environment, it is called the metagenome — and it's a common source of interest in scientific study too.

What does metagenomics add to the microbiome? ›

In conclusion, metagenomics can not only identify the diversity of the human gut microbiome, but can also reveal new genes and microbial pathways, and uncover functional dysbiosis.

What tools are used in metagenomics? ›

Metagenomics Tools
  • → 16S tools. QIIME, DADA2.
  • → Shotgun sequencing. Alignment, QC, remove host sequence, ...
  • → Assembly. MEGAHIT, ...
  • → BLAST. Blast format, E-value, FastANI, ...
  • → Bowtie2. Reference database & mapping, ...
  • → SAMtools. Coverage, consensus, SAM format, ...
  • → Ubuntu / Linux server.

What are examples of metagenomics? ›

Metagenomics has been applied to explore pharmaceutical and industrial products from environmental samples. The most popular genes that have been isolated by metagenomics were polyketide synthases (PKS) genes, which are key enzymes for synthesizing polyketide antibiotics.

What are the limitations of metagenomics? ›

The sequencing of metagenomics is facing many challenges. (1) The DNA of environmental microorganisms cannot be extracted completely. (2) Sequencing process may miss low-abundance microorganisms. (3) There is no “gold standard” for sequencing processing software.

What is next-generation sequencing and how does it work? ›

Next-generation sequencing (NGS) is a massively parallel sequencing technology that offers ultra-high throughput, scalability, and speed. The technology is used to determine the order of nucleotides in entire genomes or targeted regions of DNA or RNA.

What is next-generation sequencing microbiology? ›

Next generation sequencing (NGS) determines the DNA sequence of a complete bacterial genome in a single sequence run, and from these data, information on resistance and virulence, as well as information for typing is obtained, useful for outbreak investigation.

How does next gen seq work? ›

How NGS Works. The basic next-generation sequencing process involves fragmenting DNA/RNA into multiple pieces, adding adapters, sequencing the libraries, and reassembling them to form a genomic sequence. In principle, the concept is similar to capillary electrophoresis.

How is metagenomics different from other genomics? ›

The main difference between genomics and metagenomics is the number of organisms evaluated in an assay or sample. Genomics studies the genome of a single organism while metagenomics studies the collection of different organisms' genomes within a sample.

Which molecule is used for the analysis in a metagenomic study? ›

Metagenomic researchers isolate DNA and RNA from a sample of the habitat, such as soil or sea-water, without isolating or identifying individual organisms. The DNA or RNA is then analyzed by various genomic procedures, including shotgun DNA sequencing, PCR, RT-PCR, and so forth.

What is the advantage of studying bacterial communities using metagenomics? ›

In recent years, the use of metagenomics in the studies of soil microbial communities has enabled researchers to have an overview not only of the diversity, but also the functional traits, which are an important approach to define microbiological parameters.

Why metagenomic analysis is very much effective for non culturable cells? ›

In fact, metagenomics may provide insight into genome variation of organisms that can be readily cultured. If genetic variation in the environmental population is of interest, it may be more productive to clone the genome from the natural population than analyze the genomes of individuals cultured from it.

Which of the following correctly describes a metagenomic approach in the clinic? ›

Which of the following correctly describes a metagenomic approach in the clinic? The general strategy of metagenomics is to study a complex mixture of organisms by isolating them and studying them individually in the laboratory.

What are the application of bioinformatics in metagenomics? ›

Metagenomic approaches are now commonly used in microbial ecology to study microbial communities in more detail, including many strains that cannot be cultivated in the laboratory. Bioinformatic analyses make it possible to mine huge metagenomic datasets and discover general patterns that govern microbial ecosystems.

What makes metagenomic data different from traditional genome sequencing data? ›

In sequencing studies, unlike traditional microbial genomic sequencing projects, metagenomics research attempts to determine directly the whole collection of genes within an environmental sample (i.e., the metagenome), and analyze their biochemical activities and complex interactions.

Is metagenomics culture dependent or independent? ›

Like other molecular or serological CIDTs, the detection of microbes in metagenomics is independent of culture (Schloss and Handelsman, 2005) in contrast to WGS, which is also reliant on a pure culture (Hasman et al., 2014).

What type of information can we get from genomics metagenomics? ›

On its own, metagenomics gives genetic information on potentially novel biocatalysts or enzymes, genomic linkages between function and phylogeny for uncultured organisms, and evolutionary profiles of community function and structure.

What does metagenomic sequencing reveal? ›

Shotgun metagenomic sequencing allows researchers to comprehensively sample all genes in all organisms present in a given complex sample. The method enables microbiologists to evaluate bacterial diversity and detect the abundance of microbes in various environments.

What is metagenomic RNA sequencing? ›

Metagenomic next-generation sequencing (mNGS) is a shotgun sequencing approach in which all of the nucleic acid (DNA and RNA) in a clinical sample is sequenced at a very high depth, 10-20 million sequences per sample.

How does metagenomic approach help identify newer novel genes? ›

Metagenomics approach has been developed to identify and select microbial genes synthesizing novel molecules. This approach directly utilizes the large number of microbial genomes present in an environmental niche for example in soil in water such as ocean or in human gut.

What are the principle of metagenomics? ›

Metagenomics, the principle of which relies on the genomic analysis of a sample from a complex environment containing more than one microorganism, provides a view of the composition of this sample. Metagenomic studies became increasingly accessible with the advent of Next Generation Sequencing (NGS) [4].

What is the biggest advantage of metagenomics? ›

Like other molecular biological tools (MBTs), metagenomics eliminates the need to grow organisms in the laboratory, thus eliminating the biases associated with traditional, cultivation-based methods like plate counts.

What is metagenomics microbiome? ›

Metagenomics: towards a better understanding of the human gut microbiome. Metagenomics was first described in 1998 by Handelsman and Rodon. 35 ,36 It was defined as analysis of the collective genomes that are present in a defined environment or ecosystem, hence giving insight into functions of non-cultivated bacteria.

Why is metagenomics so important for exploring microbial diversity? ›

Metagenomics relies on isolating the genetic material from an environmental sample to determine the amount and variety of microbes present without needing to culture anything at all.

What does metagenomics add to the microbiome? ›

In conclusion, metagenomics can not only identify the diversity of the human gut microbiome, but can also reveal new genes and microbial pathways, and uncover functional dysbiosis.

What are the applications of metagenomics in health? ›

It can be used to determine gut microbial species and their abundance, and allows to monitor human health and well-being. Metagenomics sheds the light into the development of probiotics. Monitoring of human-associated bacterial communities allows to establish ways to modulate them, so as to optimize human health.

What is the difference between microbiome and metagenomics? ›

The microbiome definition in biology refers to the microorganisms and their genes whereas the microbiota only refers to the microbes themselves. If you just want to talk about all the genes in an environment, it is called the metagenome — and it's a common source of interest in scientific study too.

What is metagenomics and its applications? ›

Metagenomics is the study of a collection of genetic material (genomes) from a mixed community of organisms. In short, metagenomics is a new way to study microorganisms in a specific environment by using functional gene screening or sequencing analysis.

How do scientists use metagenomics? ›

Using metagenomics, researchers can analyze microbial diversity and also identify new proteins, enzymes, and biochemical pathways. Metagenomics has been used to identify new beneficial genes from the environment, together with novel antibiotics, enzymes that biodegrade pollutants, and enzymes that make novel products.

Why is metagenomics the most revolutionary application of genomics? ›

Metagenomics is revolutionary because it replaced the practice of using pure cultures. Pure cultures were used to study individual species in the laboratory, but did not accurately represent what happens in the environment. Metagenomics studies the genomes of bacterial populations in their environmental niche.

What are examples of metagenomics? ›

Metagenomics has been applied to explore pharmaceutical and industrial products from environmental samples. The most popular genes that have been isolated by metagenomics were polyketide synthases (PKS) genes, which are key enzymes for synthesizing polyketide antibiotics.

What are the major limitations of metagenomics? ›

The sequencing of metagenomics is facing many challenges. (1) The DNA of environmental microorganisms cannot be extracted completely. (2) Sequencing process may miss low-abundance microorganisms. (3) There is no “gold standard” for sequencing processing software.

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