Metagenomics

It was only recently that metagenomics began to be used for disease detection in suspected
outbreaks and the study of microbial populations. A small number of specialist labs were the
first to get access to diagnostic apps, which were then employed on a small number of high-
acuity patients (1, 2). There has been a widening of access due to lower costs, more availability
of sequencers, and more user-friendly data analysis tools Researchers later found that the yield
rose in dozens of individuals with negative conventional testing (6). Larger investigations in
more prevalent circumstances have lately been made possible by multiplex PCR panels, whose
methods and prices have been reduced (7). Analytical performance is often evaluated by
comparing metagenomics to the current standard of care of culture, PCR, and antigen assays.
Many patients might benefit from early detection of infections for which specialized tests (and
therapy) exist but are not requested until late or for pathogens that take days to weeks to
develop in culture. To attain its full potential, clinical metagenomics testing must be more often
and earlier in the workup, at a time when good treatment may have a significant influence on
outcomes. NGS-based procedures including fast genome sequencing for infant disease
detection, non-invasive prenatal testing, and cancer aren't widely used despite increased
evidence and demand. Analyses of clinical metagenomics performed on over 500 hospitalized
children were presented in this issue by Tao et al. (8). (274 requiring intensive care). According
to the findings, clinical validity was established as well as the necessity to improve test
performance, standardize processes, and speed result interpretation in order to make the
technology more widely used.
Metagenomics tests have a wide range of analytical performance, which varies depending on
the pathogen, the sample type, and the individual analytical technique. In the lack of reference
techniques with equal breadth and resolution, it is impossible to measure PERFORMANCE for
metagenomics tests, which may discover thousands of bacteria (whether hazardous, useful, or
of no influence on the patient). Genome sizes range from a few thousand to many millions of
base pairs (bp) and are protected by simple protein capsids, lipid membranes, or hardy cell
walls. There are over 1000 human pathogens with such diverse biological properties, such as:
single- or double-stranded RNA or DNA; single- or double-stranded RNA (9). Genetic similarity
does not always correspond with species boundaries, necessitating pathogen-specific
techniques for diagnosing infectious diseases. It is very uncommon for distinct pathogens (or
even non-pathogenic bacteria) to share >99% of their genome sequence with strains of the
same species (leading to the same diagnosis). Biochemical and taxonomic properties, thus,
represent significant and frequently unique hurdles for the extraction of nucleic acids (NA),
library construction, and identification of pathogens based on sequences, and ultimately
analytical performance. Even if analytical performance is known, it is simply a proxy for clinical
validity.
Tao et al. instead focused on clinical validity, which necessitates interpretation of laboratory,
imaging, and clinical data, by comparing findings to clinical diagnosis. The "capacity of a test to
accurately identify a patient with regard to a diagnostic, prognostic, or predictive category" is
known as clinical validity, and it is dependent on the criteria used to choose patients and the
clinical outcomes measured (10). As a result of their vulnerability to both common and rare
illnesses, children are an essential group to examine. The clinical sensitivity of metagenomics
was 74% higher than that of traditional diagnostics, however the specificity was 59% lower (38
percent and 83 percent , respectively). A critical requirement was met when sensitivity for
fungal infections was increased from 44% to 94% using conventional methods. Detection of
fungal infections in immunocompromised individuals is essential for early, focused therapy and
to avoid inappropriate use of antifungals. 53 percent of metagenomics-positive samples
included many possible pathogens, underlining the necessity of testing and interpretation. Only
197 of the 303 patients who had genuine positive findings were diagnosed with a single
bacterial infection. As determined by normalized sequencing read count, the causal pathogen
was also the most prevalent in 176 (89.3%) of these and in another 17 (8.5%), the pathogen
was second most numerous. In clinical microbiology labs, pathogens with a higher abundance in
culture are given more weight, and our findings are in line with that strategy. There were
significant advantages of in-house metagenomic testing versus send-out testing, as seen by its
quick turn-around time of 1.3 days on average.
Processes must be standardized to ensure that the findings of metagenomics research are
generalizable and to restrict the growth of the increasing number of labs undertaking clinical
metagenomics. Different pathogen extraction efficiency, sequencing depth, and read length, as
well as the specimen's NA concentration and composition (pathogen, other microbial, and
host), collection, transport, and storage, as well as data analysis databases, algorithms, and
detection thresholds, all have a significant impact on test performance. Using one approach
against another may have a major influence on extraction efficiency and yield for both the host
and the microbe. Extraction stages need to be monitored thoroughly for consistent results.
Laboratory procedures are sometimes exclusive to a certain research or diagnostic practice,
which is an unfortunate truth in today's world. Even in the same laboratory, different
techniques may be used. More than half of the samples in the research by Tao et al. were
analyzed using DNA sequencing (DNA-seq), RNA sequencing (RNA-seq) and DNA-seq plus RNA-
seq. Some diseases could only be discovered using RNA-seq in the later group, illustrating the
importance of technique selection in research.
Data analysis relies on standardization even more. Pathogen detection relies on extensive,
balanced databases that include accurately annotated reference sequences, classification
algorithms, and optimal detection/reporting criteria for each pathogen. There is a huge
disparity in the quantity of genome sequences that are available for each disease (hundreds of
thousands for some, no complete genomes for many others). Microbial sequences in public
databases need significant curation because of the high prevalence of incorrect annotation. The
majority of research are based on lab-specific databases since there are no widely agreed
standards in place. Finding the right sensitivity/specificity thresholds may be a challenge due to
the difficulty of optimizing them. It is common for infections to be identified using shotgun
metagenomics, which relies on detection of a tiny amount of their genomes. For instance, Tao
et al. examine the effect of RPM > 3 and RPM > 10 criteria (RPM, pathogen reads per million
total reads). This represents 9000 to 30000 bp of pathogen sequence (for 150 bp reads) with
around 20 million total reads per sample, or 60 and 200 pathogen reads, respectively, for a
positive result. The average bacterial genome contains less than 1% of the DNA found in
viruses. As a result of the random distribution of reads throughout the pathogen genome, their
value varies greatly depending on the region's usefulness in identifying and characterizing the
organism. As explained in this work, thresholds based on normalized read counts alone are
difficult to determine at best and sometimes inaccurate, particularly when they need to be
pathogen specific. In publications, data analysis procedures and detection criteria are typically
left out, making it difficult to comprehend and repeat the findings. The use of clinical
metagenomics will be made easier with the standardization of laboratory and data processing
procedures.

Metagenomics

Uploaded by

Copyright:

Available Formats

Metagenomics

Uploaded by

Document Information

Original Description:

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Metagenomics

Uploaded by

Copyright:

Available Formats

It was only recently that metagenomics began to be used for disease detection in suspected

You might also like