Metagenomics: A Novel Tool For Livestock & Poultry Improvement (PDF)

REVIEW ARTICLE Agricultural Reviews, Volume 44 Issue 2: 264-268 (June 2023) Metagenomics: A Novel Tool for Livestock and Poultry Improvement: A Review Sachin S. Pawar1, Pallavi A. Mohanapure2, Manoj P. Brahmane3, Mukesh P. Bhendarkar1, Avi...

REVIEW ARTICLE Agricultural Reviews, Volume 44 Issue 2: 264-268 (June 2023) Metagenomics: A Novel Tool for Livestock and Poultry Improvement: A Review Sachin S. Pawar1, Pallavi A. Mohanapure2, Manoj P. Brahmane3, Mukesh P. Bhendarkar1, Avinash V. Nirmale1, Nitin P. Kurade1 10.18805/ag.R-2167 ABSTRACT A distinct mechanism of generating metagenome is metagenomics which includes sequencing the whole DNA extracted from an environmental sample, mapping it to a reference database followed by gene annotation. Advancement in sequence-based metagenomics has significantly reduced processing costs and has acquired a rapid pace. Metagenomics has been crucial in investigating “hidden” genetic characteristics and construction biotechnological processes through the discovery of novel genes, enzymes, pathways and bioactive molecules with entirely different or improved biochemical functions. Metagenomics is a fairly recent addition to the molecular toolbox and is the simplest, impartial way of challenging the adaptive ability of microbial populations. Metagenomics is beneficial in recognising the complex consortium of bacteria, protozoa, archaea, fungi, etc. and association amongst them resulting in higher feed utilization and productivity of animals. It allows formulating probiotics feed materials, as well as in immunomodulation in both livestock and poultry. This review emphasizes significant recent achievements in metagenomics, offers insights into the possibilities, modern- day challenges and its utility in livestock and poultry. Key words: Metagenomics, Metagenome, Livestock, Sequencing. The existence of DNA molecules in a cell’s different position 1 ICAR-National Institute of Abiotic Stress Management, Baramati- indicates the chromosomal or extrachromosomal identity 413 115, Maharashtra, India. such as nuclear genome (nuclear DNA), mitochondrial 2 ICAR-Indian Agricultural Research Institute, Pusa, New Delhi-110 genome, non-chromosomal genetic elements such as 012, India. viruses, plasmids, transposable elements etc. PCR-RAPD 3 ICAR-Central Institute of Fisheries Education, Mumbai-400 061, is the dominant approach for studying the structure of the Maharashtra, India. population without specific knowledge (Sharma et al., 2004); Corresponding Author: Sachin S. Pawar, ICAR-National Institute single nucleotide polymorphism is used mainly for the study of Abiotic Stress Management, Baramati-413 115, Maharashtra, of heterogeneity by genotyping by PCR-RFLP which helps India. Email: [email protected] with economically relevant characteristics in the association field (Sahu et al., 2017). The latest concept continued as How to cite this article: Pawar, S.S., Mohanapure, P.A., metagenome which is referred to as “beyond single Brahmane, M.P., Bhendarkar, M.P., Nirmale, A.V. and Kurade, N.P. genome”. The term metagenome applies to the collective (2023). Metagenomics: A Novel Tool for Livestock and Poultry Improvement: A Review. Agricultural Reviews. 44(2): 264-268. genomes of all community members possessing a pool of doi: 10.18805/ag.R-2167. microorganisms. Metagenome entails all the genetic material found in a biological study consisting of multiple genomes Submitted: 22-01-2021 Accepted: 14-09-2021 Online: 07-10-2021 of individual organisms. Metagenomics has been described as a culture-independent, function-based or sequence- huge unexplored genetic and metabolic diversity pool. Efforts based collective Microbial Genome Analysis present in a have been made to record gut microbial diversity in birds. certain environment (Riesenfeld et al., 2004). The phrase Animal nutritionists are striving for combining current metagenomics (including the specific biological areas, knowledge of rumen activity with a potential vision on how known as ecogenomics, microbial community genomics or rumen microbiology and animal nutrition could be combined environmental genomics) to study genomes from all the with metagenomics. A better understanding of the microbes in an environment separated from the environment phenomenological mechanism of manipulating amino and controlled in vitro, as contrasted to the nucleus of one nitrogen development and absorption can enable farm organism (Handelsman et al., 1998). Metagenomics relates animal nutritionists to significantly improve nutritional to the analysis of collective genomes of the relevant nitrogen reformation into microbial protein. A broader environmental community and makes it possible to obtain understanding of the mechanistic processes that modify information about whole species of microorganisms, such amino nitrogen production and uptake can help livestock as deep oceans, soil or gut ecosystem etc. The poultry gut nutritionists enhance the overall conversion of dietary comprises millions of miscellaneous microorganisms. Most nitrogen to microbial protein. This might relay crucial details of these species remain uncharacterized and represent a to further develop mechanistic models that explain rumen 264 Agricultural Reviews  Metagenomics: A Novel Tool for Livestock and Poultry Improvement: A Review activity and analysing dietary conditions that affect the dramatically compared to the traditional Sanger sequencer. effectiveness of processing dietary nitrogen to milk protein With the emergence of newer sequencing technologies, the (Firkins et al., 2007). viability of metagenomics projects has also increased in Molecular tools to study metagenomics recent years. These newer sequencing technologies offer cheaper, quicker and higher sequencing throughputs. 16S Ribosomal RNA sequencing Illumina sequencing employs the method of sequence The occurrence of hypervariable regions within the 16S rRNA by synthesis. The sequencing process begins by ligating gene offers a species reliable signature sequence that is template DNA to an adapter chain and then to a glass flow important for bacterial phase identification. The 16S rRNA array. Bridge amplification reveals the Template DNA, which sequencing is commonly used to classify species and increases each copy to nearly 1,000 copies. Using variants in prokaryotic species and determine the isothermal polymerase and 32 inactivated fluorescent phylogenetic interactions within them. The advantages of nucleic acids, Illumina can add solitary bases to each cycle. using ribosomal RNA are that many of the cells contain Through base addition that reads the fluorescent tag is Ribosomes and ribosomal RNA and RNA genomes are followed by an imaging step. Single-base integration is of strongly conserved in nature. considerable advantage since context-specific failures, like Phylogenetic oligonucleotide microarrays (PhyloChips) those induced by homopolymers, Repetitive and low- complex regions can be sequenced conveniently over and PhyloChips rely entirely on standard molecules used by the regions of repeated and low complexity are avoided. The rRNAs and their encoding genes for taxonomic and mean error rate per sequence produced is between 1 and 2 environmental research of microorganisms. Through this percent. This output is at most ten times that of Sanger approach a sample can be analysed simultaneously with sequencing. Fault rates are well defined in the read, with thousands of rRNA (gene) targeted samples, lending in low rates at the 5 end, progressing to somewhat higher several cases an effective diagnosis of target species. A error rates at the 3 end. Errors that rely on insertion/deletion developing application of PhyloChips is the intensely parallel are exceptionally low, with such an error margin of less than study of microbial community structural feature relationships 0.01%. There are significant prejudices against G + C- or A with the aid of employing in vivo substrate-mediated isotope + T-rich sequences, more likely caused by amplification labelling of rRNA (DeSantis et al., 2007; Loy et al., 2011). processes of DNA templates (Dohm et al., 2008, Aird et al., Such probes target all 121 demarcated prokaryotic orders 2011). Since Illumina’s readings in prokaryotic genomes are and enable 8741 bacterial and archaeal taxa to be detected longer than the normal perfect match repeat period (Kassai- at almost the same time. PhyloChip technology has been Jáger et al., 2008), relatively complex metagenomes will used during bioterrorism monitoring, bioremediation, climate produce completed or almost full genomes using the Illumina change and source detection of pathogen pollution for rapid framework alone (Hess et al., 2011). identification of microbial biological populations (Brodie Real-time (SMRT) sequencing is lately developed et al., 2007; Rastogi et al., 2010). sequencing technique that has a significant impact on the Sequencing platforms for metagenomics genomics and metagenomics fields (Metzker, 2010). This Sanger sequencing or chain-terminator sequencing is one technique uses real-time single-molecule (SMRT) of the first innovations developed in sequencing technology. sequencing that is similar to single-molecule DNA Sanger sequencing quickly became the gold standard in sequencing. SMRT sequencing uses the zero-mode sequencing technology due to ease of operation and waveguide (ZMW ), through a single DNA polymerase precision. However, this method has disadvantages that enzyme is fixed as a template for a single DNA molecule to make it difficult to use in metagenomic sequencing. The the bottom of a ZMW. The ZMW is an illuminated amount of approach of chain-terminator sequencing is biologically observation small enough to allow observation of a single biased (Sorek et al., 2007) and the foreign DNA need to be DNA nucleotide (also known as a base) inserted through cloned inside a bacterial vector. Sanger sequencing is an DNA polymerase. Each of the four bases of DNA is fused to expensive and low-throughput technique. As an outcome, one of four different fluorescent dyes. Once a nucleotide is Sanger-based metagenomic projects are frequently confined inserted by DNA polymerase, which diffuses from the ZMW to fosmid sequencing and bacterial artificial chromosome detection region wherein the fluorescence is no longer libraries or microbial cultures of limited variability. accessible, the fluorescent tag is cleaved off. A detector Next-generation sequencing solved some of the identifies the fluorescent sign of nucleotide incorporation, limitations of Sanger sequencing namely, cheaper and the base call is produced according to the dye’s resulting sequencing costs per base, considerably higher throughput, fluorescence. Despite high read-length, this technique is simpler library preparation and exclusion of cloning step. restricted by a high error rate and low coverage. 96 sequence data (reads) per run are produced by Sanger Nanopore sequencing is a fourth-generation sequencer whereas Next-Generation Sequencing (NGS) can sequencing technique that employs nanopores (biological produce between one million and billion reads per run. That or solid-state) with advantages of label-free, ultra-long reads, is the key reason why NGS could increase the throughput high throughput and minimal material requirements Volume 44 Issue 2 (June 2023) 265  Metagenomics: A Novel Tool for Livestock and Poultry Improvement: A Review (Feng et al., 2015). It employs electrophoresis to move an Whenever a large number of sequences are lost during unknown sample nanopore system also contains an the denoising process, high-quality sequences result are electrolyte solution and when the continuous electrical field obtained, however, the level of stringency needed to obtain is applied, an electrical current may be detected in the system. this high quality has been debated (Gaspar et al., 2012). The nanopore dimensions and structure of the DNA or RNA Gene prediction that the nanopore occupies governs the intensity of density The process of identifying protein and RNA sequences in electrical current over a nanopore surface. A single DNA coded on the DNA sample is gene prediction/gene calling. and RNA molecule can be sequenced with the Nanopore Gene prediction can be made on post assembly contigs on sequencing without PCR amplification or chemical labelling reads from the unassembled metagenome, based on the of the sample. It has the potential to provide relatively low- applicability and performance of the assembly (Kunin et al., cost genotyping, higher test reliability and quick sample 2008). One of the most important challenges in bioinformatics analysis with the ability to demonstrate results in real-time is identifying the location of protein-coding areas using (Niedringhaus et al., 2011; Si and Aksimentiev 2017). computational approaches. Two classes of methods are Next-generation sequencing (NGS) techniques are generally adopted: similarity-based searches and ab initio widely recognized as the most effective tools for gene prediction using gene structure as a template to detect genes sequencing, giving a deep insight into the ecology of microbial (Zhou et al., 2004). controlled activities. NGS technologies are used for a variety of purposes, including single-gene targeted sequencing to Applications in livestock and poultry whole-genome sequencing and shotgun metagenome For millions of people around the world, the livestock industry sequencing. NGS is also known as high-throughput is both an economic enterprise and a survival enterprise sequencing and is used to Identify numerous modern (Karangiya et al., 2016). Meat and milk produced by ruminants environmentally sustainable RNA samples including recovery are important agricultural products and provide nutritionally of high-quality mRNA from environmental samples, brief half- rich food for humans. The livestock industry, however, faces lives of mRNA species and isolation of mRNA from other RNA major challenges due to declining natural resources and the species. It offers affordable access to the metatranscriptome resulting increase in the cost of production, as well as and allows a microbial population to profile the whole-genome environmental effects on farming ruminants (Morgavi et al., expression. Furthermore, the transcripts can also be explicitly 2013). The level of intake and digestibility of feed in livestock quantified (Carola and Daniel 2011). is related to methane production (Das 2018). Feed digestion and enteric methane production are essential functions that Denoising could be controlled by providing a detailed overview of the Denoising is important for 16S metagenomics data analysis rumen microbiome. Advances in DNA sequencing methods which is platform-specific i.e., certain systems (e.g., Illumina) and bioinformatics are shaping our perception of microbial needs less denoising than others (e.g. pyrosequencing). diverse communities like the mammalian gastrointestinal tract. Despite being computationally expensive, denoising The application of these strategies to the rumen ecosystem pyrosequencing data is important due to intrinsic errors has enabled the analysis of microbial diversity under different produced by pyrosequencing that can lead to erroneous conditions of diet and growth. The sequencing of genomes operational taxonomic units (OTUs). A technique called from many bacterial and archaeal cultured rumen species “flowgram clustering” eliminates troublesome readings and provides extensive knowledge of their physiology. Microbiome improves taxonomic analytical accuracy. Until now, several research is gaining attention in livestock products, as it helps denoising algorithms have been developed, (Reeder and to elucidate diseases and efficiency processes. The rumen Knight, 2010; Quince et al., 2011; Johanna et al., 2013; microbiota in cattle is directly associated with the digestive Balzer et al., 2013). Denoising is very effectively performed process of feed and accessibility of host nutrients and is by Amplicon-Noise (Quince et al., 2011), a tool using the considered responsible for digesting million of tons of following fundamental steps: cellulosic material worldwide to provide people with milk and meat (Hackmann and Spain, 2010). The rumen microbiota Noise reads filtering: Reads are truncated depending on rapidly digests plant material and has recently drawn the presence of low signal intensities. researchers interested in the cost-effective system for Eliminating pyrosequencing noise: The difference transforming lignocellulosic plant material into biofuel. A between the flow grams is defined and the real sequences metagenomic study using rumen as an effective cellulose and their frequencies are concluded by an expectation- fermenter fed with switchgrass and restoration of the fiber- maximization algorithm (EM). attached microbiome showed the rumen microbiota’s ability Extracting PCR noise: The same ideas are applied to to colonize and degrade biofuel biomass rapidly (Hess et al., eliminate PCR errors. 2011). Previous studies have related well known taxonomical Detection and elimination of chimera: On each sequence, groups or system composition with feed reliability or residual exact pairwise alignments are performed on all sequences feed intake (Jami et al., 2014; Jewell et al., 2015; Roehe of equal or greater excess, which is the set of probable et al., 2016). Most of these studies used 16S rRNA sequencing parents. as an outline of the microbiota. The gastrointestinal tract 266 Agricultural Reviews  Metagenomics: A Novel Tool for Livestock and Poultry Improvement: A Review microbial profiles of chicken and Guinea fowl was analysed and use of newer approaches and techniques and the using a metagenomic method to understand the microbial reduction in the cost of sequencing. Analysing gut microbial diversity of both avian organisms. For this analysis, DNA diversity would help in classifying the strains that are was collected from the chicken and Guinea fowl’s gastro- responsible for the animal’s adaptability to natural conditions. intestinal environment. The region encoding hypervariable Studying gut microbial diversity in different species of 16s rRNA was targeted to decipher the composition of livestock and poultry can help in recognizing the probiotics microbial communities in organisms using the communities with both growth-promoting and immune metagenomics approach (Palys et al., 1997). 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