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SGUL - Genomics > The Metagenome > Flashcards

The Metagenome Flashcards

1
Q

Define the following terms: Genomics, Transcriptomics, Proteomics, Metabolomics

A
  • Genomics: Whole cell gene content
  • Transcriptomics: Whole cell gene expression
  • Proteomics: Whole cell protein content
  • Metabolomics! Whole cell metabolite content
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2
Q

Why is it important to understand different organisms in an environment?

A
  • Organisms do not live in isolation
  • Most environment have a complex mixture of species
  • Many organisms cannot be cultured
  • Important to understand interactions within these complex mixtures
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3
Q

Define the term “Metagenomics”

A

The study of genetic material recovered directly from the environment samples (systems)

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4
Q

What is a micro biome?

A

A Microbial community living in a reasonably well defined habitat that has distinct physio chemical properties

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5
Q

What is Microbiota?

A

An ecological community of commensalism and pathogenic microorganisms (bacteria, archaea, protists, fungi, viruses)

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6
Q

What part of the Microbiomes are considered environmental?

A
  • Deep sea microbiome
  • Soil microbiome
  • Hospital microbiome
  • Subway microbiome
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7
Q

What part of the Microbiomes are considered human/living?

A
  • Gut microbiome
  • Skin microbiome
  • Oral microbiome
  • Vaginal microbiome
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8
Q

How does taxonomic diversity vary in the human microbiome?

A

Taxonomic diversity Varies by body site

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9
Q

What is the significance about the human microbiome?

A
  • A microbiome is unique to each individual, even between twins
  • Changes in the microbiome have been associated with multiple illnesses e.g cancer, depression
  • Individuals with Gut microbiome can classify as lean or obese with >90% accuracy
  • Early life gut microbiomes linked to development of allergic conditions
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10
Q

What are other significant factors about microbiomes?

A
  • Stool microbiome during Clostridium difficile infection (CDI) are quite different from healthy stool
  • CDI has greater effects on stool microbiome than host genetic factors
  • Faecal microbiota transplant is able to cure CDI
  • Restorwtion of the stool microbiome to that of healthy state is rapid following transplantation
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11
Q

What are the technological approaches of metagenomics?

A
  • Using Targeted PCR amplification
  • 16S rRNA, bacteria
  • internal transcribed spacer
  • Whole genome shotgun sequencing
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12
Q

What is 16S targeted PCR amplification?

A

16S ribosomal RNA is a component of 30S small subunit of prokaryotic ribosomes

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13
Q

Describe the steps involved in 16S targeted PCR amplification

A

Sample collection -> DNA extraction -> 16S PCR amplification -> Sequencing -> Analysis

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14
Q

How can the data from 16S targeted PCR amplification be analysed?

A

Using different softwares such as QIIME2, Mothur, DADA2

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15
Q

What are the high throughput sequencing machines?

A
  • Roche 454
  • Illumina HiSeq
  • IonTorrent PGM
  • Illumina MiSeq
  • PacBio
  • ONT MinION
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16
Q

Which ones are the short read sequencing?

A
  • Roche 454
  • Ion Torrent PGM
  • Illumina MiSeq
  • Illumina HiSeq
17
Q

Which ones are the long read sequencing?

A
  • PAC Bio
  • ONT MinION
18
Q

Which variable regions are chosen for the 16S targeted PCR amplification?

A
  • Phylogenetic signal
  • Amplicon length
19
Q

What are the problems with these methods?

A

They’re very sensitive to
- Contamination
- Operator
- Reagents

They’re important for low biomass samples

20
Q

Where are 16S rRNA genes found?

A
  • 16S rRNA gene found in all bacteria
  • Mitigate potential contamination
  • Randomise Samples
  • Note batch numbers of reagents
  • Sequence negative controls
21
Q

What is the problem with full length 16S sequencing?

A
  • They produce High error rates
  • Introduce noise
22
Q

What is the advantage with using full length 16S sequencing?

A
  • Increased throughput
  • Reducing cost of sequencing technologies
  • Reducing studies using 16S targeted PCR amplification
23
Q

Describe the whole genome shotgun flow

A
  • Host cell often in excess in the sample
  • No amplification step to enrich for bacterial DNA

Sample dependent, typical yields of contaminating human reads:
- Faecal <10% human reads
- Saliva, nasal, skin sample: >90% human reads

24
Q

What is one way you can enrich your sample without undergoing amplification?

A

Pre-Extraction
- Differential lysis of mammalian cells
- Enriches for intact microbial cells
- Potential bias towards gram positive bacteria

25
Q

What is another way you can enrich your sample without undergoing amplification?

A

Post- Extraction
- Enzymatic degradation of methylated nucleotides target mammalian DNA
- Bias against AT rich bacterial genomes

26
Q

Give 3 types of applications for Metagenomics

A
  • Environmental
  • Animal
  • Clinical diagnostics
27
Q

Briefly describe the applications of meta genomics in terms of environment

A

Environmental genome shotgun sequencing of the Saragossa sea
- 1.045 billion base pairs sequenced
- Elucidate the gene content, diversity and relative abundance of the organisms

28
Q

Briefly describe the applications of meta genomics in terms of environment (PART 2)

A
  • Estimated to derive from at least 1800 genomic species
  • Identified 148 previously unknown bacterial phyllotypes
  • Identified over 1.2 million previously unknown genes
29
Q

Briefly describe the applications of meta genomics in terms of Animal Microbiomes

A

Rumen Microbiome
- First of four chambers in cow’s stomach
- Contains a mix of bacteria and other organisms which ferment complex carbohydrates to produce short chain fatty acids
- Generated 6.5 Tera bases short and long read sequences from 283 ruminant cattle
- Assembled 4941 genomes including 3 whole chromosomes assemblies of rumen bacteria

30
Q

Briefly describe the applications of meta genomics in terms of Clinical meta genomics

A

Diagnostic Microbiology
- standard is to culture isolate and then identify using matrix assisted laser desorption/ionisation (MALDI)
- Many organisms can’t be cultured
- Can identify hard to culture organisms in patient samples
- Identify antibiotic resistance repertoires directly from clinical samples

31
Q

Briefly describe the applications of meta genomics in terms of Clinical meta genomics (PART 2)

A

Diagnostic microbiology (cont.)
- Potential to develop diagnostics based on differences in micro biomes

32
Q

Briefly describe the applications of meta genomics in terms of Clinical meta genomics (PART 3)

A

Public health
- Infection control and outbreak management
- Surveillance of anti microbial resistance in the food supply

SGUL - Genomics (21 decks)

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