16 s rdna sequence analysis ANITA MARGRET bhc
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Provides a basic understanding of 16S RNA sequencing which can associate to its advance involvement in metagenomics
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Assessment of microbial population diversity in polymicrobial research sample...
Assessment of microbial population diversity in polymicrobial research sample...Thermo Fisher Scientific
Analysis of 16S sequences in microbial populations using NGS gives a rapid overview of the community diversity, and is usually performed by sequencing one or two hypervariable regions (V-regions), out of the nine present in the 16S rRNA gene. In this study we compared the community structure of fecal, oral and water microbiomes by analyzing sequences from a single variable region, or from the seven V-regions (V2, V3, V4, V6-7, V8 and V9) included into Ion 16S™ Metagenomics Kit (multi-V analysis)16s
1) 16S rRNA sequencing is the gold standard for bacterial identification and can identify novel, rare, or aberrant bacterial strains that other phenotypic methods cannot.
2) The document presents data from sequencing 300 clinical isolates, identifying many new species and some new genera, and providing definitive identifications in 88% of cases.
3) While powerful, 16S rRNA sequencing has some limitations like requiring a pure culture and difficulty differentiating closely related species, and interpretation requires consideration of database issues.
SSRP Abstract
PopZ is a polar protein in Caulobacter crescentus that organizes a subcellular domain involved in chromosome segregation. This study used error-prone PCR to generate over 150 mutants of the C-terminal domain of PopZ to study its interactions with ParB and ParA, two proteins that pull chromosomes apart during cell division. Fluorescence microscopy identified mutants that affected the localization of PopZ or its interaction with ParB and ParA. The findings suggest ParB and ParA share a binding site on PopZ and the C-terminal domain of PopZ is critical for its localization, disrupting proper chromosome segregation and cell division when mutated.
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In order to identify prokaryotic species in a sample, it is often necessary to culture the sample for hours or days to increase the abundance of bacteria to assayable levels. This often precludes the rapid identification of infectious species.
Furthermore, some species are not easily culturable. We
have developed a facile research method for identifying
bacterial species by 16S ribosomal RNA sequencing on the
Ion Torrent platform. The Ion 16S™ Metagenomics Kit is
designed to PCR amplify the hypervariable regions of the 16S
gene of bacteria. We used this kit to construct libraries from
15 retrospective samples of synovial fluid with various
bacterial species either spiked in or present at collection.
Libraries were sequenced on the Ion PGM™ system and the
data analysis performed using the Ion Reporter™ workflow
which provides an automated analysis solution. Bacteria
present in the samples were correctly identified in samples
containing a single spiked-in species, mixed-species samples,
and in infected samples. Thus, the Ion Torrent™ platform
provides a mechanism for rapidly identifying bacteria that are
present in research samples without culturing.16S classifier
The diversity of microbial species in a metagenomic study is commonly assessed using 16S rRNA gene sequencing. With the rapid developments in genome sequencing technologies, the focus has shifted towards the sequencing of hypervariable regions of 16S rRNA gene instead of full length gene sequencing. Therefore, 16S Classifier is developed using a machine learning method, Random Forest, for faster and accurate taxonomic classification of short hypervariable regions of 16S rRNA sequence. It displayed precision values of up to 0.91 on training datasets and the precision values of up to 0.98 on the test dataset. On real metagenomic datasets, it showed up to 99.7% accuracy at the phylum level and up to 99.0% accuracy at the genus level. 16S Classifier is available freely at http://metagenomics.iiserb.ac.in/16Sclassifier and http://metabiosys.iiserb.ac.in/16Sclassifier.
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This document discusses plasmids and bacteriophages. It defines plasmids as small, extrachromosomal DNA molecules that can replicate independently of chromosomal DNA within bacterial cells. Plasmids are classified based on their transmissibility and function. Methods for purifying plasmid DNA from bacterial cells are also described. Bacteriophages are viruses that infect bacterial cells, and the document discusses the genetic organization and life cycles of important bacteriophages such as lambda. The roles of plasmids and bacteriophages in genetic engineering and molecular cloning are also summarized.
Mammalian artificial chromosome
Human artificial chromosomes (HACs) could be useful for gene therapy by allowing large gene fragments to be stably introduced and expressed in target cells without disrupting existing genes. HACs are small, artificial microchromosomes that can carry exogenous DNA and act as entirely new chromosomes in human cells. They were first constructed in 1997 by adding alpha-satellite DNA to telomeric and genomic DNA. There are two accepted methods for creating HAC vectors - altering a natural chromosome or de novo construction of a novel chromosome, though the latter has proven more difficult. HACs are preferable to other transgenic methods like yeast/bacterial artificial chromosomes due to their separation from the original genome, which provides greater stability and prevents insert
Recommended
Assessment of microbial population diversity in polymicrobial research sample...
Assessment of microbial population diversity in polymicrobial research sample...Thermo Fisher Scientific
Analysis of 16S sequences in microbial populations using NGS gives a rapid overview of the community diversity, and is usually performed by sequencing one or two hypervariable regions (V-regions), out of the nine present in the 16S rRNA gene. In this study we compared the community structure of fecal, oral and water microbiomes by analyzing sequences from a single variable region, or from the seven V-regions (V2, V3, V4, V6-7, V8 and V9) included into Ion 16S™ Metagenomics Kit (multi-V analysis)16s
1) 16S rRNA sequencing is the gold standard for bacterial identification and can identify novel, rare, or aberrant bacterial strains that other phenotypic methods cannot.
2) The document presents data from sequencing 300 clinical isolates, identifying many new species and some new genera, and providing definitive identifications in 88% of cases.
3) While powerful, 16S rRNA sequencing has some limitations like requiring a pure culture and difficulty differentiating closely related species, and interpretation requires consideration of database issues.
SSRP Abstract
PopZ is a polar protein in Caulobacter crescentus that organizes a subcellular domain involved in chromosome segregation. This study used error-prone PCR to generate over 150 mutants of the C-terminal domain of PopZ to study its interactions with ParB and ParA, two proteins that pull chromosomes apart during cell division. Fluorescence microscopy identified mutants that affected the localization of PopZ or its interaction with ParB and ParA. The findings suggest ParB and ParA share a binding site on PopZ and the C-terminal domain of PopZ is critical for its localization, disrupting proper chromosome segregation and cell division when mutated.
Rapid 16S Next Generation Sequencing for Bacterial Identification in Polymicr...
Rapid 16S Next Generation Sequencing for Bacterial Identification in Polymicr...Thermo Fisher Scientific
In order to identify prokaryotic species in a sample, it is often necessary to culture the sample for hours or days to increase the abundance of bacteria to assayable levels. This often precludes the rapid identification of infectious species.
Furthermore, some species are not easily culturable. We
have developed a facile research method for identifying
bacterial species by 16S ribosomal RNA sequencing on the
Ion Torrent platform. The Ion 16S™ Metagenomics Kit is
designed to PCR amplify the hypervariable regions of the 16S
gene of bacteria. We used this kit to construct libraries from
15 retrospective samples of synovial fluid with various
bacterial species either spiked in or present at collection.
Libraries were sequenced on the Ion PGM™ system and the
data analysis performed using the Ion Reporter™ workflow
which provides an automated analysis solution. Bacteria
present in the samples were correctly identified in samples
containing a single spiked-in species, mixed-species samples,
and in infected samples. Thus, the Ion Torrent™ platform
provides a mechanism for rapidly identifying bacteria that are
present in research samples without culturing.16S classifier
The diversity of microbial species in a metagenomic study is commonly assessed using 16S rRNA gene sequencing. With the rapid developments in genome sequencing technologies, the focus has shifted towards the sequencing of hypervariable regions of 16S rRNA gene instead of full length gene sequencing. Therefore, 16S Classifier is developed using a machine learning method, Random Forest, for faster and accurate taxonomic classification of short hypervariable regions of 16S rRNA sequence. It displayed precision values of up to 0.91 on training datasets and the precision values of up to 0.98 on the test dataset. On real metagenomic datasets, it showed up to 99.7% accuracy at the phylum level and up to 99.0% accuracy at the genus level. 16S Classifier is available freely at http://metagenomics.iiserb.ac.in/16Sclassifier and http://metabiosys.iiserb.ac.in/16Sclassifier.
Artificial chromosomes - YAC and BAC
This document discusses yeast artificial chromosomes (YACs) and bacterial artificial chromosomes (BACs). YACs are engineered chromosomes derived from yeast DNA that can clone very large DNA sequences in yeast cells of up to 1 megabase. BACs are cloning vectors derived from bacterial DNA that can clone DNA fragments of up to 300 kilobases in E. coli. Both systems allow cloning and propagation of large DNA fragments, but YACs can hold more DNA while BACs are more stable and better for functional analysis in mammalian cells.
Plasmids and bacteriophages
This document discusses plasmids and bacteriophages. It defines plasmids as small, extrachromosomal DNA molecules that can replicate independently of chromosomal DNA within bacterial cells. Plasmids are classified based on their transmissibility and function. Methods for purifying plasmid DNA from bacterial cells are also described. Bacteriophages are viruses that infect bacterial cells, and the document discusses the genetic organization and life cycles of important bacteriophages such as lambda. The roles of plasmids and bacteriophages in genetic engineering and molecular cloning are also summarized.
Mammalian artificial chromosome
Human artificial chromosomes (HACs) could be useful for gene therapy by allowing large gene fragments to be stably introduced and expressed in target cells without disrupting existing genes. HACs are small, artificial microchromosomes that can carry exogenous DNA and act as entirely new chromosomes in human cells. They were first constructed in 1997 by adding alpha-satellite DNA to telomeric and genomic DNA. There are two accepted methods for creating HAC vectors - altering a natural chromosome or de novo construction of a novel chromosome, though the latter has proven more difficult. HACs are preferable to other transgenic methods like yeast/bacterial artificial chromosomes due to their separation from the original genome, which provides greater stability and prevents insert
artificial chromosome
Artificial chromosomes are engineered DNA molecules that can stably maintain large DNA fragments like natural chromosomes. There are four main types: yeast artificial chromosomes, bacterial artificial chromosomes, human artificial chromosomes, and mammalian artificial chromosomes. Yeast and bacterial artificial chromosomes are used to clone large DNA fragments up to 500kb and 30kb respectively. Human and mammalian artificial chromosomes are engineered to act as entirely new chromosomes in human and other mammalian cells, carrying exogenous genes for applications like gene therapy.
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Yeast artificial chromosomes (YACs) are engineered yeast chromosomes containing megabase-sized fragments of foreign DNA. YACs are created by ligating bacterial plasmid DNA containing yeast centromeres, telomeres, and replication origins to fragments of genomic DNA. This creates an artificial yeast chromosome capable of replicating inside yeast cells and accommodating very large DNA inserts of up to 2 megabases. YACs can be used to generate whole genome libraries for mapping genes and identifying chromosomal sequences important for constructing mammalian artificial chromosomes. However, YACs are less stable than bacterial artificial chromosomes and prone to rearrangements due to their large insert sizes.
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The document discusses minichromosome technology in plants. It begins by defining a minichromosome as an extremely small version of a chromosome that carries genes and transfers genetic information autonomously. It then discusses how minichromosomes can be produced through telomere truncation or from pre-existing B chromosomes. The behavior of minichromosomes during cell division and meiosis is explained. Methods for manipulating genes on minichromosomes through site-specific recombination and gene stacking are also summarized.
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1. Cosmid vectors are hybrid vectors derived from plasmids that contain the cos site from bacteriophage lambda, allowing them to clone DNA fragments up to 40 kb in size.
2. Yeast artificial chromosomes (YACs) are engineered yeast chromosomes that can clone very large DNA fragments, averaging 200-500 kb but up to 1 MB, taking advantage of yeast cell machinery.
3. Bacterial artificial chromosomes (BACs) are DNA constructs based on fertility plasmids that can clone up to 300 kb fragments and address issues with YAC stability and recombination.
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Function-based analysis
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Examples for metagenomics projects
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DNA contains the genetic code that determines an organism's traits. It is made up of nucleotides with four bases (A, T, G, C) that pair up in a double helix structure. Genes, located on chromosomes, are segments of DNA that encode instructions for making proteins. DNA is passed from parents to offspring through sexual reproduction, which involves the fusion of egg and sperm cells to form a new individual with a random combination of the parents' genes. Mutations can occur in DNA and be inherited, sometimes causing genetic disorders.
DNA_and_inheritance.ppt
DNA contains the genetic code that determines an organism's traits. It is made up of nucleotides with four bases - adenine, guanine, cytosine, and thymine - that pair up in a double helix structure. Genes, located on chromosomes, are segments of DNA that encode instructions for making proteins. DNA is passed from parents to offspring, and mutations can occur that cause genetic disorders or diseases. The human genome project mapped the entire human DNA sequence, consisting of 3 billion base pairs organized into 22,000 genes on 23 chromosome pairs.
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Microbiology has experienced a transformation during the last 25 years that has altered microbiologists' view of microorganisms and how to study them. The realization that most microorganisms cannot be grown readily in pure culture forced microbiologists to question their belief that the microbial world had been conquered. We were forced to replace this belief with an acknowledgment of the extent of our ignorance about the range of metabolic and organismal diversity.
Thesis
A 70-year-old man was hospitalized with bacteremia and acute cholecystitis. Blood and gallbladder samples grew gram-positive rods that were identified as Lactobacillus salivarius using 16S rRNA gene sequencing, but various phenotypic methods gave inconsistent or incorrect results. The study evaluated using 16S rRNA gene sequencing for identifying gram-positive rods in the clinical laboratory, finding it more accurate and able to identify organisms that phenotypic methods could not.
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This document discusses various methods for identifying and characterizing prokaryotes, including phenotypic and genotypic characteristics. Identification using phenotypic characteristics involves microscopic analysis, staining techniques like Gram stain, culture characteristics on selective media, and biochemical tests of metabolic differences. Genotypic identification uses techniques like nucleic acid probes, PCR, sequencing ribosomal RNA genes. Characterizing strain differences can be done through biochemical and serological typing, genomic typing methods like PFGE and ribotyping, phage typing, and antibiograms.
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Biotechnology: Yeast Artificial Chromosome Cloning Vector
Yeast artificial chromosomes (YACs) are engineered yeast chromosomes containing megabase-sized fragments of foreign DNA. YACs are created by ligating bacterial plasmid DNA containing yeast centromeres, telomeres, and replication origins to fragments of genomic DNA. This creates an artificial yeast chromosome capable of replicating inside yeast cells and accommodating very large DNA inserts of up to 2 megabases. YACs can be used to generate whole genome libraries for mapping genes and identifying chromosomal sequences important for constructing mammalian artificial chromosomes. However, YACs are less stable than bacterial artificial chromosomes and prone to rearrangements due to their large insert sizes.
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The document discusses minichromosome technology in plants. It begins by defining a minichromosome as an extremely small version of a chromosome that carries genes and transfers genetic information autonomously. It then discusses how minichromosomes can be produced through telomere truncation or from pre-existing B chromosomes. The behavior of minichromosomes during cell division and meiosis is explained. Methods for manipulating genes on minichromosomes through site-specific recombination and gene stacking are also summarized.
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1. Cosmid vectors are hybrid vectors derived from plasmids that contain the cos site from bacteriophage lambda, allowing them to clone DNA fragments up to 40 kb in size.
2. Yeast artificial chromosomes (YACs) are engineered yeast chromosomes that can clone very large DNA fragments, averaging 200-500 kb but up to 1 MB, taking advantage of yeast cell machinery.
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INTRODUCTION
HISTORY
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Sequence-based analysis
Function-based analysis
Application of metagenomics
Future Directions of metagenomics
Examples for metagenomics projects
Dna and genes
DNA contains the genetic code that determines an organism's traits. It is made up of nucleotides with four bases (A, T, G, C) that pair up in a double helix structure. Genes, located on chromosomes, are segments of DNA that encode instructions for making proteins. DNA is passed from parents to offspring through sexual reproduction, which involves the fusion of egg and sperm cells to form a new individual with a random combination of the parents' genes. Mutations can occur in DNA and be inherited, sometimes causing genetic disorders.
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DNA contains the genetic code that determines an organism's traits. It is made up of nucleotides with four bases - adenine, guanine, cytosine, and thymine - that pair up in a double helix structure. Genes, located on chromosomes, are segments of DNA that encode instructions for making proteins. DNA is passed from parents to offspring, and mutations can occur that cause genetic disorders or diseases. The human genome project mapped the entire human DNA sequence, consisting of 3 billion base pairs organized into 22,000 genes on 23 chromosome pairs.
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Microbiology has experienced a transformation during the last 25 years that has altered microbiologists' view of microorganisms and how to study them. The realization that most microorganisms cannot be grown readily in pure culture forced microbiologists to question their belief that the microbial world had been conquered. We were forced to replace this belief with an acknowledgment of the extent of our ignorance about the range of metabolic and organismal diversity.
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This document discusses various methods for identifying and characterizing prokaryotes, including phenotypic and genotypic characteristics. Identification using phenotypic characteristics involves microscopic analysis, staining techniques like Gram stain, culture characteristics on selective media, and biochemical tests of metabolic differences. Genotypic identification uses techniques like nucleic acid probes, PCR, sequencing ribosomal RNA genes. Characterizing strain differences can be done through biochemical and serological typing, genomic typing methods like PFGE and ribotyping, phage typing, and antibiograms.
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Complications of wound healing like infection, hyperpigmentation of scar, contractures, and keloid formation.
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إضغ بين إيديكم من أقوى الملازم التي صممتها
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تتميز هذهِ الملزمة بعِدة مُميزات :
1- مُترجمة ترجمة تُناسب جميع المستويات
2- تحتوي على 78 رسم توضيحي لكل كلمة موجودة بالملزمة (لكل كلمة !!!!)
#فهم_ماكو_درخ
3- دقة الكتابة والصور عالية جداً جداً جداً
4- هُنالك بعض المعلومات تم توضيحها بشكل تفصيلي جداً (تُعتبر لدى الطالب أو الطالبة بإنها معلومات مُبهمة ومع ذلك تم توضيح هذهِ المعلومات المُبهمة بشكل تفصيلي جداً
5- الملزمة تشرح نفسها ب نفسها بس تكلك تعال اقراني
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كل التوفيق زملائي وزميلاتي ، زميلكم محمد الذهبي 💊💊
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16 s rdna sequence analysis ANITA MARGRET bhc
- 1. 16S rDNA Sequence Analysis
- 2. • What are the characteristics of 16S rRNA that make it • useful as an analyte for bacterial identification? • • Ubiquitous • • Highly Conserved Molecule • • Contains variable and hyper-variable regions of sequence • • Extensively studied and represented in database
- 4. STRUCTURES • •Primary Structure • RNA sequence • Secondary Structures • Formation of double • stranded regions (helices) • Tertiary Structures • Folding of 1o and 2o structures • Quaternary Structures • Interactions with other • molecules (e.g. RNAs, proteins)
- 5. Small Subunit rRNA 16S in Eubacteria 18S in Eukaryotes E.coli 16S rRNA Molecule Typically used for comparison
- 6. Why Mycobacteria? • • Slow growing and fastidious • • Phenotypic identification can take 2 to 8 weeks • • Cultural data: Growth rate, pigmentation and morphology • • Accu-probe testing: Can test for M.tuberculosis, MAC, • M.kansasii and M.gordonae only. • •• • Biochemical tests: Tween 80 hydrolysis, arylsulfatase etc. • •• • HPLC, GLC, TLC: for lipid wall analysis
- 8. Precious Time is Wasted • Patients might receive inappropriate therapy. • Point mutations can confer antimicrobial resistance • Atypical Mycobacteria respond differently to standard • antimicrobials
- 9. Other Gene Targets •Hsp 65 : 65 kDa gene which is also highly conserved • Highly specific for Mycobacteria •recA gene
- 12. Pilot Study • • 22 different cultures of microorganissms subjected to • 16S rDNA sequence analysis • • • • Results compared to phenotypic data • • • • Includes several Mycobacterium cultures
- 13. Methods Outline • •Isolate bacterial DNA • •Amplify 16S rRNA gene • •Sequence a portion of 16S rRNA gene (Region 1) • •Compare sequence obtained with GenBank to find “Match”
- 15. Compare to GenBank • •Gen Bank is a freely available web-based database • of 16S rDNA sequences • •Contains bacteria, fungi and other microrganisms • •BLAST “match” was done
- 16. Advantages • •Prevents misidentification by phenotypic analysis • •Early diagnosis • •Validated by numerous studies • •Not subject to false-negative results • •Can be matched against GenBank, RIDOM and the ribosomal database library • •Identification of unusual isolates
- 17. Drawbacks • •Ambiguous data in databases: base errors, incomplete sequences • •Difficult to differentiate some organisms like • M. chelonae and M. abcessus
- 18. Clinical Relevance • •Early detection of microrganisms • •Needs very little substrate to work with • •Better targeting of antimicrobials • •Used successfully by Sacchi et al in the recent bioterrorism outbreak for early detection of B. anthracis • •Incorporated into routine diagnostics at a number of centers