Metagenomics - Potentials And Limitations

Introduction

The microorganisms that live inside or on human impacts not only health but also are related to metabolism, skin health, mental health, and diseases such as asthma, allergy, inflammatory bowel disease, cardiovascular disease, cancers, infections, and even autoimmune disorders. Metagenomics is the latest approach to studying total microorganisms' nucleotide sequences. It is the sum of the genomes of microorganisms in environmental samples. Microorganisms are obtained from the land, sea, and from extreme environments. Marine organisms are large in number than soil microorganisms. Latest sequencing technologies, such as third-generation sequencing (TGS) and next-generation sequencing (NGS), are used. Metagenomics is used in the research field of biology, agriculture, energy environment, pollution control, etc.

What Is Metagenomics?

Metagenomics is also called the microbial environmental genome, which is the study of the form and function of the nucleotide sequence of the total microbes which usually live on the surface of human skin or in an environment such as water or soil. It involves the analysis of single-subunit rRNA (ribosomal RNA) genes from many samples, and this data is useful in studying taxonomic profiles of microorganisms from the sample. This study is more comprehensive than marker gene approaches. This study focuses on studying microorganisms' diversity, relationship and interaction with the environment, evolutionary and genetic relationship, community constitutes, and functional activities.

What Are The Steps In Metagenomics?

Metagenomics involves the following steps:

• DNA Extraction: The first step in metagenomics is the extraction of DNA (deoxyribonucleic acid) from the environmental sample. To improve the identification of target sequences in organisms, unwanted sequences should be removed.

• Sequencing: Next-generation sequencing techniques, such as Illumina's short-read sequencers [e.g., HiSeq (high-throughput sequencer) and NextSeq (next-generation sequencer)], are commonly used in metagenomics studies. The sequencing data is collected based on standard molecular biology protocols.

• Read Depth Determination: The read depth is determined based on the taxonomic profile of the target organism(s) of interest. If the goal is to identify a single pathogen, it may require less depth.

• Taxonomic Profiling: After sequencing, the obtained reads are processed to assign taxonomic identities to the organisms present in the sample. Tools like Kraken (a bioinformatics tool and software application used in metagenomics for taxonomic classification of DNA sequences obtained from environmental samples) are commonly used for taxonomic profiling.

• Functional Analysis: In addition to taxonomic information, metagenomics also aims to understand the functional capabilities of the microbial community. This is achieved by analyzing the protein-coding genes present in the metagenomic data. Databases like KEGG (Kyoto encyclopedia of genes and genomes) are used for functional annotation. Tools like MG-RAST (metagenome rapid annotation using subsystem technology) and MEGAN (MEtaGenome ANalyzer) are commonly employed for functional composition analysis.

• Metabolic Reconstruction: Once the functional composition is determined, the next step is to reconstruct the metabolic pathways and interactions within the microbial community. Tools like HUMAnN (HMP Unified Metabolic Analysis Network) are utilized for metabolic reconstruction of metagenomic data.

What Are The Potentials Of Metagenomics?

Metagenomics has applications in the medical field, such as the recognition of microorganisms in the intestinal tract and infections of the bloodstream, central nervous system, or lungs. They are useful in studying antibiotic-resistant genes and bacteria. Potentials of metagenomics include:

• Pathogen Identification: Pathogens from the sample, in particular those which are hard to culture, can be identified with the help of metagenomics.

• Discovering Enzymes: It has the potential to discover new enzymes derived from microbial metabolism.

• Controlling And Preventing Environmental Pollution: Metagenomic studies of microorganisms in water can help in the removal of biological nitrogen and phosphorus. The functional genes in these microorganisms can help in the degradation of plastics, pesticides, plastics, petroleum hydrocarbons, polycyclic aromatic hydrocarbons, and other organic pollutants. Microbial electrochemical systems make use of electroactive bacteria to recover electrical energy from organic wastes.

• Diagnosis Of Microbial Diseases: The diagnosis of microbial disease traditionally includes culture-based methods but has limitations like failure in isolation of disease-causing organisms, being time-consuming, and requiring a large amount of labor. But with the help of metagenomics, which has the potential to characterize and identify several viral and bacterial pathogens with the advantages of reducing cost and high sensitivity. It can face challenges like requiring multiple complex steps. A sequence subtraction method called pathSeq and clinical pathoScope, which is much faster, helps in the removal of human DNA by leaving a small number of datasets to search. Sequence-based ultrarapid pathogen identification is used to diagnose patients with encephalitis (caused by a virus).

• Studying Of Dysbiosis: Metagenomics can help in studying dysbiosis, which is the imbalance of the gut microbiome and a complex disease such as clostridium difficile infection, which occurs due to dysbiosis. Clostridium difficile infection (CDI) occurs due to overuse of antibiotics which causes a reduction in the gut microbiome and colonization resistance with increased growth of clostridium difficile. The treatment should include antibiotics that kill the pathogen and spare the host microbial microflora. Clinical treatment of CDI includes Metronidazole and Vancomycin, which are harmful to gut microbiota. According to a metagenomic study,

  1. The microbiome’s functional state plays an important role in a single species.

  2. Fecal microbiome transplantation (FMT) has shown a high success rate.

  3. Patients with recurrent CDI have high primary bile acids, and the use of FMT can restore normal microbiota and bile composition and can inhibit microbiota.

  4. Butyrate, caused by some gut microbiota, can prevent CDI.

• Studying Antimicrobial Resistance: The metagenome contains resistome, which is the collection of resistance determinants among the microbial community, which can help in predicting the resistance patterns and is useful in the case of known resistance mechanisms of a particular community. According to metagenomics:

  1. The microbial imbalance of the gut is the main cause of vancomycin-resistant enterococci.

  2. Mykrobe predictor software helps in identifying the alleles which are associated with antibiotic resistance.

What Are The Limitations Of Metagenomics?

The limitations of metagenomics include:

• Extraction of genes with less abundant microorganisms is impossible.

• There can be a loss of DNA fragments during the gene cloning process.

• Because of limited screening methods, all the enzyme screening requirements cannot be fulfilled.

• A small number of newly discovered enzymes can be used due to limitations in PH and temperature.

• Only a few positive clones can be obtained from tens of thousands of clones.

• Heterologous expression of foreign genes frequency is low.

• The gold standard for sequencing processing is not yet developed.

• Because of the imperfect microbial database, sequencing data cannot be analyzed.

• Pathogenic organisms which are found in this study can be dead or dormant (inactive).

• The accuracy of metagenomics requires verification by large sample data.

• A false-negative result can occur in case of intracellular bacterial infection because of incorrect methods of cell wall rupture.

• For pathogen diagnosis, it is recommended that metagenomics should not be used first; instead, PCR (polymerase chain reaction) or immunoassay should be done.

• Before data analysis, it is required to remove DNA contamination from the environmental microbes and humans.

Conclusion

Therefore metagenomics is helpful in the identification of antibiotic resistance genes, microbial dysbiosis, diagnosis, and the management of disorders that are related to viral, bacterial, and fungal organisms. It has limitations like limited screening methods, can obtain only a few clones, and requires verification by large sample data.