LUBNA SHAKOOR M-PHIL “I” ADVANCES IN MICROBIAL   GENETICS
PROKARYOTIC TRANSCRIPTOMICS AND METAGENOMICS
 
TRANSCRIPTOMICS <ul><li>Transcriptomics or gene expression profiling is the study of complete set of RNA transcripts produ...
BACKGROUND <ul><li>Previously Prokaryotic transcriptomics was underestimated due to reasons such as: </li></ul><ul><li>Sim...
TECHNIQUES  FOR  TRANSCRIPTOMIC  <ul><li>RNA Sequencing </li></ul><ul><li>Tilling Arrays </li></ul>
RNA-Seq <ul><li>Whole Transcriptome Shotgun Sequencing ”WTSS”. </li></ul><ul><ul><li>Sequencing cDNA. </li></ul></ul><ul><...
RNA Enrichment Techniques
Wang  et al.  2009
 
Mapping <ul><li>Place reads onto a known genomic scaffold </li></ul><ul><ul><li>Requires known genome and depends on accur...
DNA microarrays <ul><li>DNA microarrays  rely on the  hybridization  properties of nucleic acids to monitor DNA or RNA abu...
Gene expression assays <ul><li>The main types of gene expression assays: </li></ul><ul><ul><li>Serial analysis of gene exp...
Applications of microarrays <ul><li>Measuring transcript abundance (cDNA arrays); </li></ul><ul><li>Genotyping; </li></ul>...
Tiling Array <ul><li>Genome array consisting of overlapping probes </li></ul><ul><li>Finer Resolution </li></ul><ul><li>Be...
Tiling Arrays http://en.wikipedia.org/
Tiling Array http://en.wikipedia.org/
Affymetrix’s Photolithographic Approach GeneChip A Dan Nettleton, Department of Statistics,  IOWA STATE UNIVERSITY, Copyri...
Source: www.affymetrix.com
Source: www.affymetrix.com
Source: www.affymetrix.com
Affymetrix GeneChip ® Affymetrix GeneChip ® Limits: 1: 100,000 transcripts ~ 5 transcripts/cell
 
What is Metagenomics? <ul><li>Traditional microbial genomics </li></ul><ul><ul><li>Sequence the genome of one organism at ...
Techniques in Metagenomics <ul><li>Isolate DNA </li></ul><ul><ul><li>Depends on sample type </li></ul></ul><ul><li>Clone D...
From Figure 2 in Daniel, R. (2005) &quot;The Metagenomics of Soil&quot; Nature Reviews Microbiology 3: 470-478.
Analysis of Metagenomics Data <ul><li>Metagenomes are big </li></ul><ul><ul><li>Soil has as many as 40,000 individual micr...
Understanding Microbial Communities <ul><li>Some questions metagenomics may answer </li></ul><ul><ul><li>Are certain adapt...
CONCLUSION <ul><li>“ After billions of years of evolution, prokaryotes have developed a huge diversity of regulatory mecha...
REFERENCES <ul><li>http://www.nugo.org /transcriptomics </li></ul><ul><li>http://bioinformatics-made-easy.blogspot.com/201...
 
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PROKARYOTIC TRANSCRIPTOMICS AND METAGENOMICS

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After billions of years of evolution, prokaryotes have developed a huge diversity of regulatory mechanisms, many of which are probably uncharacterized. Now that the powerful tool of whole-transcriptome analysis can be used to study the RNA of bacteria and archaea, a new set of un expected RNA-based regulatory strategies might be revealed.

Metagenomics, together with in vitro evolution and high-throughput screening technologies, provides industry with an unprecedented chance to bring biomolecules into industrial application.

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  • Arial Traditional genomics focuses on the sequencing and analysis of the genomes of individual organisms. When applied to microbes, it typically involves culturing the organism of interest followed by sequencing. Metagenomics is a new area of microbial genomics that aims to sequence the full or partial genomes of all members of a microbial community (also called a consortium). The term microbial community refers to the complex microbial ecosystems that exist almost everywhere in nature. For example, a project in soil metagenomics might extract DNA from a soil sample in a corn field and attempt to sequence all the DNA found in the sample. By directly sequencing the DNA, researchers bypass the need to culture organisms. Since only a very small minority of single-cell organisms have been successfully cultured in the laboratory, metagenomics becomes a very powerful technique for sequencing genes from organisms that can not be cultured. Alternatively, homologous genes from a variety of organisms in the microbial community can be selectively sequenced via PCR using tags that exist in known organisms.
  • Arial Isolating DNA from an environmental sample is the first step of any metagenomics study. Different types of samples often require specialized extraction techniques; however, once the DNA is extracted most metagenomics projects will take one of several approaches. A schematic of the techniques used to study the soil metagenome is shown in the slide. Once DNA is isolated it is cloned, entered into some kind of vector (bacterial artificial chromosome, plasmid, cosmid, etc.), and then inserted into an appropriate bacterial host. The bacteria can then be used in functional screens for specific types of biochemical activity ( e.g. proteases). Alternatively, DNA from the metagenome can be used for sequencing. In some cases this can even result in the complete or almost complete genomes of individual species in the environmental sample.
  • Arial The greatest challenge in studying metagenomes is their size. It is estimated that as many as 40,000 distinct species of microorganisms exist in a single soil sample, making the soil metagenome orders of magnitude larger than the human genome. Present sequencing technology makes it impossible to fully sequence that amount of DNA in a reasonable time or at a reasonable expense (though this may change in the not so distant future). To make matters worse, not all organisms are equally abundant in microbial communities, meaning that even greater amounts of DNA would have to be sequenced in order to obtain adequate coverage of rarer genomes. Nonetheless, significant progress has been made in studying metagenomes. For example, functional screens of metagenomic sequences have uncovered novel genes for antibiotics, antibiotic resistance, lipases, and proteases to name only a few. Phylogenetic studies have revealed the broad distributions of species within a particular microbial community. In some cases, it has even been possible to fully sequence the most common members of a particular community.
  • Arial Though metagenomics is still in its infancy, it holds great promise for answering fundamental questions about the structure and dynamics of microbial communities. The slide poses a small sample of such questions. Metagenomics has already yielded several interesting discoveries. For example, a study of ocean surface water uncovered a new class of rhodopsin genes in alpha-proteobacteria. Rhodopsins are proteins that respond to light and serve a range of purposes in a wide variety of organisms, including the detection of light in the retinal cells of humans and other animals. Further studies of the newly discovered bacterial rhodopsins found that the light response of the proteins was tuned to match that of the light that was reaching the alpha-proteobacteria at different ocean depths. Metagenomics can also be used to observe the interactions of individual members of microbial communities. One such example is shown in the slide where fluorescence in situ hybridization (FISH) was used to visualize archaeal and bacterial species in an ocean sediment community. Bacteria are shown in green and archaea in red.
  • PROKARYOTIC TRANSCRIPTOMICS AND METAGENOMICS

    1. 2. LUBNA SHAKOOR M-PHIL “I” ADVANCES IN MICROBIAL GENETICS
    2. 3. PROKARYOTIC TRANSCRIPTOMICS AND METAGENOMICS
    3. 5. TRANSCRIPTOMICS <ul><li>Transcriptomics or gene expression profiling is the study of complete set of RNA transcripts produced by genome. </li></ul>For the better understanding of gene structures and RNA-based regulation in any organism, Transcriptomics acts as a power tool.It gives us solutions : 1. to understand genes 2. pathways involved in biological processes In short it examines the expression level of mRNAs .
    4. 6. BACKGROUND <ul><li>Previously Prokaryotic transcriptomics was underestimated due to reasons such as: </li></ul><ul><li>Simplicity as compared to Eukaryotes </li></ul><ul><li>mRNA enrichment challenges </li></ul><ul><li>But now, due to the advent of Next generation Sequencing Technologies like: </li></ul><ul><li>RNA sequencing </li></ul><ul><li>Genomic Arrays </li></ul><ul><li>We can easily interrogate whole Prokaryotic Transcriptome. </li></ul>
    5. 7. TECHNIQUES FOR TRANSCRIPTOMIC <ul><li>RNA Sequencing </li></ul><ul><li>Tilling Arrays </li></ul>
    6. 8. RNA-Seq <ul><li>Whole Transcriptome Shotgun Sequencing ”WTSS”. </li></ul><ul><ul><li>Sequencing cDNA. </li></ul></ul><ul><ul><li>Using NexGen technology. </li></ul></ul><ul><li>Revolutionary Tool for Transcriptomics. </li></ul><ul><ul><li>More precise measurements. </li></ul></ul><ul><ul><li>Ability to do large scale experiments with little starting material. </li></ul></ul><ul><ul><li>How different alleles of a gene are expressed. </li></ul></ul><ul><ul><li>Detect post-transcriptional mutations e.g., gene fusion detection. </li></ul></ul>
    7. 9. RNA Enrichment Techniques
    8. 10. Wang et al. 2009
    9. 12. Mapping <ul><li>Place reads onto a known genomic scaffold </li></ul><ul><ul><li>Requires known genome and depends on accuracy of the reference </li></ul></ul>http://en.wikipedia.org/
    10. 13. DNA microarrays <ul><li>DNA microarrays rely on the hybridization properties of nucleic acids to monitor DNA or RNA abundance on a genomic scale in different types of cells. </li></ul>Print-tip head
    11. 14. Gene expression assays <ul><li>The main types of gene expression assays: </li></ul><ul><ul><li>Serial analysis of gene expression (SAGE); </li></ul></ul><ul><ul><li>Short oligonucleotide arrays (Affymetrix); </li></ul></ul><ul><ul><li>Long oligonucleotide arrays (Agilent); </li></ul></ul><ul><ul><li>Fibre optic arrays (Illumina); </li></ul></ul><ul><ul><li>cDNA arrays (Brown/Botstein)*. </li></ul></ul>
    12. 15. Applications of microarrays <ul><li>Measuring transcript abundance (cDNA arrays); </li></ul><ul><li>Genotyping; </li></ul><ul><li>Estimating DNA copy number (CGH); </li></ul><ul><li>Determining identity by descent (GMS); </li></ul><ul><li>Measuring mRNA decay rates; </li></ul><ul><li>Identifying protein binding sites; </li></ul><ul><li>Determining sub-cellular localization of gene products; </li></ul>
    13. 16. Tiling Array <ul><li>Genome array consisting of overlapping probes </li></ul><ul><li>Finer Resolution </li></ul><ul><li>Better at finding RNA in the cell </li></ul><ul><ul><li>mRNA </li></ul></ul><ul><ul><ul><li>Alternative splicing </li></ul></ul></ul><ul><ul><ul><li>Not Polyadenylated </li></ul></ul></ul>
    14. 17. Tiling Arrays http://en.wikipedia.org/
    15. 18. Tiling Array http://en.wikipedia.org/
    16. 19. Affymetrix’s Photolithographic Approach GeneChip A Dan Nettleton, Department of Statistics, IOWA STATE UNIVERSITY, Copyright © 2008 Dan Nettleton mask mask mask mask mask mask mask mask A A C C G G T T T T T A A C C
    17. 20. Source: www.affymetrix.com
    18. 21. Source: www.affymetrix.com
    19. 22. Source: www.affymetrix.com
    20. 23. Affymetrix GeneChip ® Affymetrix GeneChip ® Limits: 1: 100,000 transcripts ~ 5 transcripts/cell
    21. 25. What is Metagenomics? <ul><li>Traditional microbial genomics </li></ul><ul><ul><li>Sequence the genome of one organism at a time </li></ul></ul><ul><ul><li>Use cultures to isolate microbe of interest </li></ul></ul><ul><li>Metagenomics </li></ul><ul><ul><li>Extract sequence data from microbial communities as they exist in nature </li></ul></ul><ul><ul><li>Bypass the need for culture techniques </li></ul></ul><ul><ul><ul><li>Sequence all DNA in sample </li></ul></ul></ul><ul><ul><ul><li>Select DNA based on universal sequences </li></ul></ul></ul>© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
    22. 26. Techniques in Metagenomics <ul><li>Isolate DNA </li></ul><ul><ul><li>Depends on sample type </li></ul></ul><ul><li>Clone DNA </li></ul><ul><li>Insert into plasmid </li></ul><ul><li>Develop sample library </li></ul><ul><li>Screen or sequence </li></ul>© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
    23. 27. From Figure 2 in Daniel, R. (2005) &quot;The Metagenomics of Soil&quot; Nature Reviews Microbiology 3: 470-478.
    24. 28. Analysis of Metagenomics Data <ul><li>Metagenomes are big </li></ul><ul><ul><li>Soil has as many as 40,000 individual microbial species </li></ul></ul><ul><ul><li>Soil metagenome orders of magnitude bigger than human genome </li></ul></ul><ul><li>Analyzing the metagenome </li></ul><ul><ul><li>Screens </li></ul></ul><ul><ul><li>Phylogenetic studies </li></ul></ul><ul><ul><li>Sequencing uncultivated organisms </li></ul></ul><ul><ul><li>Studying metagenome under different conditions </li></ul></ul>
    25. 29. Understanding Microbial Communities <ul><li>Some questions metagenomics may answer </li></ul><ul><ul><li>Are certain adaptations observable across environmental gradients? </li></ul></ul><ul><ul><li>How do different species interact? </li></ul></ul><ul><ul><li>Can lateral gene transfer be detected? </li></ul></ul>© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 From Figure 2 in Schleper, C., et al. (2005) &quot;Genomics studies of uncultivated Archaea&quot; Nature Reviews Microbiology 3: 479-488. Permission for figure 2 granted by K.Knittel and T.Loesekann,MPI Bremen, www.mumm-research.de .
    26. 30. CONCLUSION <ul><li>“ After billions of years of evolution, prokaryotes have developed a huge diversity of regulatory mechanisms, many of which are probably uncharacterized. Now that the powerful tool of whole-transcriptome analysis can be used to study the RNA of bacteria and archaea, a new set of un expected RNA-based regulatory strategies might be revealed.” </li></ul><ul><li>www.nature.com/reviews/genetics </li></ul><ul><li>“ Metagenomics, together with in vitro evolution and high-throughput screening technologies, provides industry with an unprecedented chance to bring biomolecules into industrial application.” </li></ul><ul><li>www.nature.com/reviews/micro </li></ul>
    27. 31. REFERENCES <ul><li>http://www.nugo.org /transcriptomics </li></ul><ul><li>http://bioinformatics-made-easy.blogspot.com/2010/01/bioinformatics-transcriptomics . </li></ul><ul><li>http://en.wikipedia.org </li></ul><ul><li>Prokaryotic transcriptomics: a new view on regulation, physiology and pathogenicity By Rotem Sorek and Pascale Cossart.Nature Reviews Genetics | AOP, published online 24 November 2009; doi:10.1038/nrg2695 </li></ul><ul><li>Metagenomics and industrial applications By Patrick Lorenz and Jürgen Eck, 2005. </li></ul>

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