Metagenomics in the Era of Next Generation Sequencing

Metagenomics is defined as the study of total genomic DNA obtained directly from the environmental samples, such as soil, sea water, and desert, without culturing microbes in the laboratory. Metagenomics, therefore, helps to study the microbes that are uncultivable by standard culture techniques, allowing the discovery of novel microbial species. This makes metagenomics an indispensable tool for the understanding of microbial communities. In a functional aspect, metagenomics can be used to understand the evolution, ecology, and metabolism of the microbes present in an ecological niche as well as to investigate the relationship between microbial diversity and ecosystem functioning.

Metagenomics is defined as the study of total genomic DNA obtained directly from

the environmental samples, such as soil, sea water, and desert,

without culturing microbes in the laboratory

Earlier, Sanger sequencing was used to study the microbial communities in diverse environments. Sanger sequencing is based on the integration of chain-terminating dideoxynucleotides in DNA during DNA replication. Although this method provided many new insights into these microbial communities, it suffered from major drawbacks including high cost and the inability to detect rare microorganisms. Next generation sequencing (NGS) technologies have revolutionized the field of metagenomics by allowing faster preparation of sequencing libraries and parallel sequencing of millions of sequences at low costs and improved accuracy. Several NGS platforms with different principles and features are available for metagenomics studies. These include Genome analyzer (Illumina), 454 (Roche), Ion torrent (Life Technologies), and Sequencing by Oligonucleotide Ligation and Detection (SOLiD, Life Technologies), where sequencing is based on reversible dye terminator, pyrosequencing, proton detection, and ligation, respectively. All of these NGS-based studies involve a few common steps including sample collection, DNA extraction, library synthesis, sequencing, and read pre-processing followed by data analysis and functional binning. However, these platforms differ in terms of their yields, read lengths, run times, costs, and error rates.

Metagenomics can be performed with

two different NGS-based approaches:

targeted metagenomics and shotgun metagenomics

Metagenomics can be performed with two different NGS-based approaches: targeted metagenomics and shotgun metagenomics. Targeted metagenomics, used for the profiling of microbial communities, analyzes a specific genomic region mostly through polymerase chain reaction (PCR)-based directed sequencing. Generally, the small and large subunits of ribosomal RNA genes are used as the markers for the identification of microbial species. On the other hand, shotgun metagenomics provides information regarding the microbes present in the community as well as the biological function of the community. It is based on random shearing of the total DNA into multiple short sequences followed by their sequencing and reconstruction into a consensus sequence.

NGS technologies have made a significant contribution to metagenomics by encouraging researchers to explore the microbial world. NGS has highlighted the effects of agriculture management system, resource availability, and soil stratification on microbial diversity. NGS provided the indications that the composition, diversity, and structure of the microbial community in the soil are affected by land use patterns, tree species, and soil pH. It helped in the identification of genes involved in mineral phosphate solubilization and phytic acid utilization in rhizosphere soil. NGS also identified the abundance of several microbial species in different regions of the mangrove sediments. NGS can be applied to study plant-microbe interactions and to answer several interesting questions related to interactions in rhizospheres and phyllosphere, responses to pathogen attack, and climate change. NGS technologies have also been used for the understanding of the microbial processes that can affect water quality such as algal bloom and pathogen dissemination. Additionally, metagenomics can also be applied to gain information related to the human microbiome.

Metagenomics can be integrated with

metatranscriptomics and metaproteomics

to answer broader questions related to microbial ecology

With the advent of NGS technologies in metagenomics, huge amounts of data are generated at a remarkable rate and there is an unmet need for handling and managing these huge datasets. We need to develop databases, computing systems, and algorithms to manage the data. For data deposition in the repositories, an acceptable standardized format needs to be developed that can help in the comparison of metagenomics data across different laboratories. To solve the problems associated with low genomic coverage and short read lengths, we can combine metagenomics with single cell genomics for a more accurate segregation of the metagenomic sequences into individual genomes. Metagenomics can  be integrated with metatranscriptomics and metaproteomics to answer broader questions related to microbial ecology. Compared to Sanger sequencing, NGS technologies are prone to higher error rates. These errors can be reduced during the sequencing process itself or post-sequencing by application of correction algorithms. It is recommended to use high-quality DNA and proper sequence coverage to reduce the risks of errors and artifacts. As with any study, it is critical to perform multiple biological and technical replicates for the correct interpretation of the results. The relative comparisons between microbial communities can be performed to nullify the effects of technical variations on the accuracy of results. More microbial strains need to be sequenced and characterized to increase the size of the microbial database that will help to reduce the number of sequences that represent orphan or unknown genes during metagenomics analyses.

With the decrease in cost and increase in accuracy,

NGS techniques can be applied to investigate many

significant research questions in microbial ecology

Further advances are expected in the field of metagenomics with the development of single molecule real-time (SMRT, Pacific Biosciences) sequencing which can provide improved coverage, read length, and accuracy. With the decrease in cost and increase in accuracy, NGS techniques can be applied to investigate many significant research questions in microbial ecology. Metagenomics analysis conducted at different time points can be used to elucidate general patterns and laws that govern the interactions between microbes and their environment. As the environmental samples contain DNA from both microbes and large organisms, a combinatorial analysis of these two types of DNA can provide information on the interactions between microbes and large organisms. In the future, it will be exciting and fascinating to see major breakthroughs in the field of metagenomics and its applications in microbial ecology.

This blog is based on an essay submitted for the essay writing competition 2017, organized by We The Microbiologist and Bioclues Organization.

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Author: Isha Verma

Isha Verma, PhD is currently a postdoctoral fellow at the Indian Institute of Science, Bangalore, India. Her research work is focussed on the generation, characterization, and enrichment of neural cells from mouse pluripotent stem cells. She also works as a freelance science editor at Cactus Communications and scientific consultant at Kolabtree. She loves reading, traveling, and stargazing. Follow her on Linkedin.

Editors: Rajamani Selvam and Dolonchapa Chakraborty

Rajamani Selvam is currently a Neuroscience Ph.D. student at the University of Connecticut Health, Farmington, CT. Her research focuses on understanding the interactions between growth factors and endocannabinoids in modulating acute synaptic transmission in the brain. Post-graduation, she is interested in pursuing a career in medical communications. She is passionate about communicating STEM education and outreach to middle and high schoolers. She is also a mentor for 1000 girls 1000 futures program, New York Academy of Sciences.  Away from science, she is an artist and enjoys leisure travel. Follow her on LinkedIn.

Illustrator: Bhrugu Yagnik

Bhrugu Yagnik is a Postdoctoral Fellow at Emory Vaccine Centre, Yerkes National Primate Research Centre, Emory University, Atlanta, GA and works on the development of a HIV/AIDS vaccine. His doctoral research focused on development of vaccines against Shigella using food grade Lactococcus lactis as an antigen delivery vehicle. Bhrugu has many awards to his credit. He is passionate about communicating science in creative ways. In his free time, Bhrugu indulges himself into the spirituality where he attempts to bring amalgamation of science and spirituality. Follow him on Twitter or connect with him on LinkedIn or ResearchGate.

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