WFP.PK: Molecular Methods biotechPPT1.pptx

for further study

1. MOLECULAR METHODS Assignment 01 Submitted to: dr. azhar Rashid (Professor & Chairman) Submitted by: Umeed Malik (PhD 1 st student)

2. Metagenomics Metagenomics is the study of Metagenomes. Genetic material removed directly from environmental samples. (Intro:1998) It is a study of structure and function of entire nucleotide sequences isolated and analyzed from all the organisms (typically microbes) in a bulk sample. It is often used to study a specific community of micro-organisms. Such as those residing on Human skin, in the soil or in water sample . Metagenomics is also termed as Environmental Genomics, Eco-Genomics and Community Genomics. Metagenomics is Direct Genetic Analysis (DGA) of genomes with an environmental sample. Metagenomics is an interdisciplinary field of Genetics includes the study of environmental samples using Genetic techniques.

3. Metagenomics is the study of genetic material recovered directly from environment samples,

4. Principle 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).

5. Metagenomics : Principle

6. 3 steps to metagenomics study B a sically, metagenomic studies include three important steps, 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.

7. Metagenomic techniques Metagenomics is defined as the direct genetic analysis of genomes contained with an environmental sample. The field initially started with the cloning of environmental DNA, followed by functional expression screening , was then quickly complemented by direct random shotgun sequencing (sequencing is a laboratory technique for determining the DNA sequence of an organism's genome) of environmental DNA.

8. Purpose To characterize uncultivable (not suitable for cultivation) microbes in various environments Because this relatively new field of genetic research enables studies of organisms that are not easily cultured in a Laboratory. To determine phylogenetically (A way that relates to the evolutionary development and diversification of a species or group of organisms: Phylogenetically related fish species) relevant genes.

9. Methodology Metagenomics is the genomic analysis of microorganisms by direct extraction and cloning of DNA from their natural environment. Protocols have been developed to capture unexplored microbial diversity to overcome the existing barriers in estimation of diversity .

10. Example in research and outcomes Metagenomics, developed in the first decade of the twenty-first century, laid the foundation of the “-omics” techniques and revolutionized research in microbial ecology ( Handelsman , 2004). Environmental metagenomic libraries have proved to be great resources for new microbial enzymes and aMetagenomic techniques, however, are based on the concept that the DNA retrieved directly from environmental samples theoretically represents entire community genomes which could be sequenced and analyzed in the same way as that of a whole genome of a pure bacterial culture, thus enabling microbial ecologists to glean a comprehensive understanding of whole communities.ntibiotics with potential applications in biotechnology, medicine, and industry ( Riesenfeld et al., 2004). Essentially, genetic fingerprinting techniques do not provide information on diversity beyond the selected genes that are being amplified and consequently, are useful only for partial community analysis.

11. Metagenomic Applications

12. Meta transcriptomics Meta transcriptomics is the science that studies gene expression of microbes with natural environmental i -e, The Meta transcriptome. It also allows to obtain whole gene expression profiling of complex microbial communities.

13. Principle Metatranscriptomics informs us of the genes that are expressed by the community as a whole. With the use of functional annotations of expressed genes, it is possible to infer the functional profile of a community under specific conditions, which are usually dependent on the status of the host.

14. Purpose To profile the whole genome expression of microbial communities. Metatranscriptomics has been applied to many plant-microbe interactions studies as it is able to characterize members of a microbiome that are responsible for specific functions and elucidate genes that drive the relationship of the microbiome with its host.

15. Methodology Metatranscriptomic sequencing provides direct access to culturable and non-culturable microbial transcriptome information by large-scale, high-throughput sequencing of transcripts from all microbial communities in specific environmental samples.

16. Example in research and outcomes Human gut microbiome A metatranscriptomics analysis measuring the functional activity of the gut microbiome reveals insights only partially observable in metagenomic functional potential, including disease-linked observations for IBD. IBD Inflammatory bowel disease.

17. Metaproteomic Metaproteomic or community proteomics, environmental proteomics is an umbrella term for experimental approaches to study all proteins in microbial communities and microbiomes from environment sources . The study of the structure and function of proteins, including the way they work and interact with each other inside cells

18. Principle The most common metaproteomics workflow consists of sample collection and preservation, cellular lysis, protein extraction, tryptic digestion of proteins into peptides, peptide separation by LC, and analyses of peptide masses (MS) and their fragments (MS/MS) by mass spectrometry.

19. Purpose To identify proteins produced by complex microbial communities. Through connecting genomic and metabolic information, metaproteomics is an essential approach for understanding how microbiomes function in space and time. To provides deep insights into the biodiversity of microbial communities and the complex functional interplay between microbes and their hosts or environment.

20. Methodology The most common metaproteomics workflow consists of sample collection and preservation, cellular lysis, protein extraction, tryptic digestion of proteins into peptides, peptide separation by LC, and analyses of peptide masses (MS) and their fragments (MS/MS) by mass spectrometry . Metaproteomics is a study used to qualitatively and quantitively analyze proteins in microbial communities, thus revealing microbial phenotypic information at the molecular level.

21. Example in research and outcomes To understand the roles that microorganisms play in diverse environments such as the open ocean or the human intestinal tract, we need an understanding of their metabolism and physiology. A variety of methods such as metagenomics and metaproteomics exist to assess the metabolism of environmental microorganisms based on gene content and gene expression.

22. Metabolomics Metabolomics is the large scale study of small molecules, commonly known as metabolites, within cells, biofluids, tissues or organisms. Collectively, these small molecules and their interactions

23. Principle Metabolomics is the study of metabolome within cells, biofluids, tissues, or organisms to comprehensively identify and quantify all endogenous and exogenous low-molecular-weight (<1 kDa ) (1,000 Daltons). Dalton (Da) is an alternate name for the atomic mass unit, and kilodalton ( kDa ) is 1,000 Daltons. Thus a peptide with a mass of 64kDa has a molecular weight of 64,000 grams per mole) small molecules/metabolites in a biological system in a high-throughput manner.

24. Purpose To identify and quantify metabolites To illustrate quorum sensing . To identify what the genotype of an organism its physiology and environment (what the organism eats or breaths). To offers an opportunity to look at genotype-phenotype and genotype environtype relationships. Metabolites are ultimate result of cellular pathways (changes in genome, transcriptome , proteome as well as metabolic influences).

25. Example in research and outcomes Metabolomics is an objective lens to view the complex nature of how physiology is linked to external events and conditions, as well as measure its response to perturbations such as those associated with disease. Metabolomics is the study of endogenous and exogenous metabolites in biological systems, which aims to provide comparative semi-quantitative information about all metabolites in the system. Metabolomics is an emerging and potentially powerful tool in ophthalmology research .

26. References Kleiner , M., Dong, X., Hinzke , T., Wippler , J., Thorson, E., Mayer, B., & Strous , M. (2018). Metaproteomics method to determine carbon sources and assimilation pathways of species in microbial communities. Proceedings of the National Academy of Sciences, 115(24), E5576-E5584. https://dornsife.usc.edu/labs/laketyrrell/metagenomics/ https://www.google.com/search?q=metagenomics+applications&tbm=isch&ved=2ahUKEwiv_ufEwJz8AhX-idgFHUDRAeMQ2-cCegQIABAA&oq=metagenomics+applications&gs_lcp=CgNpbWcQARgAMgUIABCABDIHCAAQgAQQGDoGCAAQCBAeUIQDWJY4YPNIaABwAHgAgAGcCYgBtSqSAQc1LTEuMy4ymAEAoAEBqgELZ3dzLXdpei1pbWfAAQE&sclient=img&ei=D1CsY--6BP6T4t4PwKKHmA4&bih=789&biw=1600#imgrc=6VpY3zcqdaXyCM http:// www.envirobiotechjournals.com/EEC/vol28issue1/EEC-1.pdf https ://www.bgi.com/us/sequencing-services/rna-sequencing-solutions/transcriptome-sequencing / http:// www.matrixscience.com/pdf/2019WKSHP2.pdf https://www.diversigen.com/ https://www.google.com/search?q=metaproteomics&sxsrf=ALiCzsYNp2xidwbLh8WdfeLOyfLV1ic-wQ%3A1672294949658&ei=JTKtY-_ wJ7_gz7sP7rKR0Ag&oq=metapro&gs_lcp=Cgxnd3Mtd2l6LXNlcnAQARgDMgQIABBDMgQIABBDMgQIABBDMgUIABCABDIFCAAQgAQyBQgAEIAEMgUILhCABDIFCAAQgAQyBQgAEIAEMgUIABCABDoKCAAQRxDWBBCwAzoHCAAQsAMQQzoECCMQJzoECC4QQzoFCAAQkQI6BwgAELEDEENKBAhBGABKBAhGGABQ2QVYjjFggkloAXABeACAAYIFiAHKK5IBAzUtOZgBAKABAcgBCsABAQ&sclient=gws-wiz-serp https:// www.slideshare.net/berciyalgolda1/metatranscriptomics.