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Systems Biology > L1-4: Overview of Systems Biology and Bioinformatics > Flashcards

L1-4: Overview of Systems Biology and Bioinformatics Flashcards

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1
Q

Every individual harbours ____ genetic variant sites.

A

4-5 million

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2
Q

Transcriptome

A

The full set of RNA molecules in one cell or a population of cells (i.e. expressed genes).

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3
Q

The aim of transcriptomic experiments is to identify ______.

A

differentially expressed genes

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4
Q

The Human Cell Atlas project aim

A

identify (based on transcriptome) and locate every cell in the human body

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5
Q

Proteomics can be distinguished into which four main aspects?

A

Sequence
Structural
Functional and interaction
Expression

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6
Q

Genomics

A

Study of the function and structure of a genome

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7
Q

Genome

A

The complete set of all genes, regulatory sequences and non-coding regions within an organism’s DNA

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8
Q

Contig

A

A set of overlapping DNA segments that together represent a consensus region of DNA.

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9
Q

Isolation of total genomic DNA - steps

A
  1. Mechanical disruption of cells/tissues (homogeniser, bead beater)
  2. Lysis of host cells (detergents such as SDS)
  3. Separation of DNA through enzymatic digestion of proteins, absorption to and release of the DNA from a chromatographic matrix (resin) and deproteinisation of the DNA solution with organic solvents (phenol / chloroform)
  4. Precipitation of DNA with ethanol or isopropanol
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10
Q

Construction of a shotgun library

A
  1. Collections of short segments of DNA generated by digestion of genomic DNA with restriction enzymes (representing the entire genome) are ligated into vector plasmids.
  2. Millions of different recombinant molecules are generated and these are propagated in bacteria or yeast.
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11
Q

Sanger Sequencing

A

When DNA binds dideoxynucleotides, they arrest DNA sequencing
The dideoxynucleotides for each of the four bases can each have a different fluorescent label so the 4 reactions can be run in the same tube.
The reaction is run on a polyacrylamide gel and fluorescence detected by an automated sequencing machine.

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12
Q

Next-generation WGS sequencing techniques

A

Illumina sequencing
Roche 454
Ion Torrent

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13
Q

Illumina Sequencing

A

100-150bp reads are used
Fragments are ligated to adapters and annealed to a slide. PCR is carried out and copies are separated into single strands.
The slide is flooded with nucleotides and DNA polymerase
An image is taken: in each read location there will be a fluorescent signal indicating the base that has been added
Terminators are removed, allowing the next base to be added. The process is repeated, adding one nucleotide at a time with imaging in between.

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14
Q

Roche 454 sequencing

A
  • DNA is fragmented, adapters added, annealed to beads and amplified by PCR. Each bead is placed in a single well of a slide.
  • The slide is flooded with one of the four nucleotides. The addition of each nucleotide releases a light signal.
  • The NTP mix is washed away and the next NTP mix is added and the process is repeated, cycling through the four NTPs.
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15
Q

Ion Torrent: Proton/PGM Sequencing

A
  • Ion Torrent does not make use of optical signals. The basis of ion torrent sequencing relies on the addition of a dNTP releasing a H+ ion.
  • DNA is fragmented, adapters added and one molecule is placed on a bead and amplified by PCR. Each bead is placed in a single well of a slide. The slide is flooded with one of the four dNTPs.
  • The pH is detected in each well. The release of a H+ ion will decrease the pH.
  • The dNTPs are washed away and the process is repeated, cycling through the dNTPs.
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16
Q

Methods for long sequence reads

A

Nanopore technology

SMRT sequencing

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17
Q

Nanopore technology

A
  • A protein nanopore is set in an electrically resistant polymer membrane.
  • An ionic current is passed through the nanopore by setting a voltage across this membrane.
  • If an analyte passes through the pore or near its aperture, this event creates a characteristic disruption in current
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18
Q

SMRT sequencing

A

Based on DNA replication:
A fluorescent label on the terminal phosphate of the dinucleotides can be detected when DNA polymerase incorporates the nucleotide into the DNA

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19
Q

The two most commonly used high-throughput methods of measuring the transcriptome are:

A

microarrays and RNA sequencing

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20
Q

Advantages of RNA-Seq over microarrays

A

Comprehensive; microarrays require known sequences and an annotated genome.
Microarrays only reveal information about ORFs.
RNA-seq covers entire genome
Detects novel transcripts
Identifies structural variations (gene fusions and alternative splicing)

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21
Q

Illumina, Roche454 and Iontorrent generate ____ bp reads

A

100

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22
Q

RNAseq workflow

A
  1. Library preparation: isolation of RNAs and generation of cDNA, selection of fragment size, and addition of linkers
  2. Illumina paired read sequencing
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23
Q

Experimental considerations of designing RNAseq experiments

A

Library construction:
choosing the population of RNA to use
Sequencing depth (required read number and coverage)
Number of technical and biological replicates

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24
Q

Majority of the RNA in a cell is ____

A

ribosomal RNA

25
Q

RNAseq library construction workflow

A
  1. Choosing the population of RNA to use
  2. Generation of strand-specific library that retains the orientation of the original RNA transcript (allows identification of anti-sense transcripts or non-coding DNA
  3. Single or paired end reads (more confidence over intron splice sites)
26
Q

Why is RNAseq sequencing depth important?

A

Adequate read depth is required to detect low-expression transcripts

27
Q

Why is the number of technical and biological replicates important in RNAseq?

A

Increase in biological replicates significantly increases the number of differentially regulated genes expressed

28
Q

Chromatography

A

The separation of components in a mixture that involves passing the mixture dissolved in a mobile phase through a stationary phase. The analyte is separated from other molecules based on differing partitioning

29
Q

Mass spectrometry

A

Identification is based on the mass spectrum comparison against standard mass spectrum from other libraries.

30
Q

GC/LC-MS generates a _______.

A

Extracted Ion Chromatogram (EIC)

31
Q

Differences between GC and LS mass spectrometry

A

LC-MS detects a wider arrange of metabolites, as well as hydrophilic and hydrophobic metabolites. However it’s less reliable and stable than GC-MS.
GC-MS is more favourable due to:
- High peak capacity
- Excellent repeatability
- Vast and readily-available electron ionised compound libraries, making compound identification easier.

32
Q

Illumina sequencing workflow

A
  1. Sample preparation; fragmentation of DNA and addition of adaptors
  2. Cluster growth
  3. Sequencing using fluorescence optics
  4. Imaging
33
Q

Variability in an organoid expression can be due to

A

Changes in expression within cell types

Changes in the proportion of cell within the organoid

34
Q

Differences between eukaryotes and prokyaryotes

A
  • DNA is linear in eukaryotes, circular in prokaryotes.
  • DNA is associated with histones in eukaryotes, naked in prokaryotes
  • Prokaryotes do not have introns
  • Prokaryotic DNA is located in the cytoplasm (no nucleus)
  • Prokaryotes don’t have organelles
  • Prokaryotes - 70S ribosomes; eukaryotes 80S
  • Prokaryotes reproduce through binary fission (asexual)
  • Prokaryotic DNA is haploid
35
Q

Prokaryotic genome size

A

Usually less than 5Mb

36
Q

Human genome size

A

3400 Mb

37
Q

PacBio RS II vs. Illumina NGS

A

PacBio RS II has higher error rates but results in a complete/closed genomic map

38
Q

Multiplex sequencing

A

Allows large numbers of libraries to be pooled and sequenced simultaneously during a single run of a high throughput instrument.
Individual barcode sequences are added to each DNA fragment that can be identified and sorted

39
Q

FASTQ format output by line

A

LINE 1: Identifier
Line 2: Sequence
Line 3: +
Line 4: Quality score in ASCII encoded format

40
Q

Examples of genome annotation pipelines

A

RAST, NCBI Prokaryotic Genome Annotation Profile; BASys

41
Q

Protein purification techniques

A

Chromatography based techniques

42
Q

Protein analysis tecnniques

A

ELISA, Western blotting, Protein microarray

43
Q

Protein characterisation techniques

A

Mass spectrometry, gel-based approaches

44
Q

Protein sequence analysis techniques

A

Edman sequencing

45
Q

Protein quantification techniques

A

ICAT, SILAC, iTRAQ

46
Q

Protein structural analysis techniques

A

NMR spectroscopy, x-ray crystallography

47
Q

Primary metabolites

A

Distributed within all living organisms and are intimately essential life processes and include ubiquitous compounds

48
Q

Secondary metabolites

A

Have only restricted distributions and are often a specific characteristic of individual organisms and species.
May not directly participate in growth and development but influence ecological interactions

49
Q

Advantages of analysing the metabolome

A
  1. The metabolome is the downstream product of gene expression so it reflects the functional level of the cell more directly.
  2. Changes in the metabolome are generally amplified relative to the proteome and transcriptome.
  3. It is estimated that metabolomics experiments are lower in costs compared to other ‘omics’ as they produce more information per experiment.
50
Q

How does NMR work?

A

Magnetic field is applied, molecules absorb and emit when placed in a strong magnetic field
Signal is proportional to the number of H in a molecule

51
Q

NMR can be used in metabolomics to

A

Accurately quantify metabolites in a complex sample relative to a spiked internal standard.
It has low sensitivity therefore needs lots of sample

52
Q

LC-MS is used for which metabolites

A

Higher molecular mass or lower thermostability metabolites

53
Q

What is unique about C. hepaticus compared to other Campylobacter species?

A

Unlike other Campylobacter species, C. hepaticus has glucose and polyhydroxybutyrate metabolism pathways. These genes may play a role in stress response in C. hepaticus and are putative virulence factors

54
Q

C. hepaticus HV10 is predicted to be able to

biosynthesise most amino acids, except

A

L-cystein and L-lysine

55
Q

RNAseq comparative transcriptomic analysis beween in vivo colonisation and in vitro conditions steps

A
  1. RNA isolation
  2. RNAseq
  3. Read mapping to C. hepaticus HV10 complete genome
  4. Differential analysis of the two conditions
  5. Predict putative virulence genes in C. hepiaticus
56
Q

Metabolomics in food microbiology

A

Identification and quantification of microbial metabolites in food e.g. Early detection of food pathogens and food spoilage microorganisms

57
Q

Proteomics in food microbiology

A

Identification and quantification of microbial proteins within a food matrix e.g. Identification of bioactive peptides and proteins which are nutritionally important

58
Q

Transcriptomics in food microbiology

A

discover the functions of food microorganisms e.g. Identify candidate genes involved in resistance by studying the differentially expressed genes under the antibiotic cultivation condition etc

Systems Biology (4 decks)

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