Marine genomics, tools and applications

Question 1

Gene definition

Answer 1

a fragment of DNA that codes for a specific protein

Question 2

Genome definition

Answer 2

all genetic information contained in an organism

Question 3

Transcriptome

Answer 3

set of all RNA molecules produced in a cell or tissue at a given time

Question 4

proteome

Answer 4

full complement of proteins expressed by the genome of cell, tissue or an organism at a specfic time point

Question 5

what happens during Translation?

Answer 5

During translation, ribosomal subunits assemble together on the strand of mRNA, where they proceed to attract tRNA molecules tethered to amino acids (circles).

A long chain of amino acids emerges as the ribosome decodes the mRNA sequence into a polypeptide, or a new protein.

Question 6

Central Dogma theory

Answer 6

a theory stating that genetic information flows only in one direction, from DNA, to RNA, to protein, or RNA directly to protein.

Question 7

DNA –> RNA

Answer 7

TRANSCRIPTION

Question 8

RNA –> PROTEIN

Answer 8

TRANSLATION

Question 9

PROTEIN –> METABOLITE

Answer 9

ENZYMATIC REACTION

Question 10

Sanger Sequences

Answer 10

incorporation of chain-terminating dideoxynucleotides (ddNTPs) by DNA polymerase during in vitro DNA replication.

Question 11

When to use Sanger sequencing?

Answer 11

variant screening studies when the total number of samples is low

Question 12

Next generation sequencing (NGS)

Answer 12

Similar principle to Sanger sequencing- The genomic strand is fragmented, and the bases in each fragment are identified by emitted signals

Question 13

NGS techniques

Answer 13

Combines steps for separation, sequencing and detection

High-throughput and speed: millions of reactions in parallel.

three steps- library preparation, amplification and sequencing

Question 14

Advantages of NGS over Sanger Sequencing

Answer 14

increased sample size
increased sequencing speed
reduced cost
increased accuracy

Question 15

NGS- isolate nucleic acids

Answer 15

confirm yield, purity and quality of nucleic acids

Question 16

NGS Library preparation

Answer 16

nucleic acids fragmented into shorter sequences and adapters attached to the sequences to ensure compatibility with the sequencing platform.

Can include PCR amplification

Question 17

NGS Data analysis

Answer 17

Read processing- base calling, QC, removal of adaptors Sequence analysis- alignment, assembly, annotation, gene counts, differential expression etc

Question 18

genomics applications

Answer 18

genome evolution
adaptive evolution
Biodiversity: Identification of species (e.g. barcoding, metagenomics etc.)

Phylogenetic relationship between taxa.

Population structure and species distribution.

Relationship between community structure and ecological function.

Question 19

Transcriptomics applications

Answer 19

Identifying mechanisms underpinning physiological functions

Environmental monitoring and early warning system for:
– Chemicals (i.e. pollutants)
– Toxins (e.g. HABs)
– Environmental change: temperature, ocean acidification, hypoxia, nutrient depletion etc.

Question 20

qPCR uses

Answer 20

quantitive real time PCR is used to quantify expression levels of a gene of interest in a given cell or tissue

Question 21

what does a qPCR require?

Answer 21

previous knowledge of gene sequence (at least partial)

requires specific primers

Question 22

qPCR mechanism

Answer 22

1- heat makes DNA unzip (95 degrees)

2- polymerase enzyme adds nucleotides (72 degrees)

3-primers stick to target gene (~55 degrees)

4- two copies of gene from one strand of DNA

Question 23

what does qPCR measure?

Answer 23

measures product accumulation in every cycle by measuring fluorescence

• determines cycle when fluorescence is fists detected
• determines cycle when fluorescence crosses a threshold set in the linear range

Question 24

Limitations of qPCR and microarrays

Answer 24

Reliance upon existing knowledge about the genome sequence.

High background levels owing to cross- hybridisation (microarrays).

Limited dynamic range of detection.

Comparing expression levels across different experiments is often difficult.

Question 25

qPCR advantages over microarrays

Answer 25

– A priori knowledge of the genome or genomic features is not required for a discovery driven
approach.

– It offers single-nucleotide resolution, making it possible to detect related genes (or features), alternatively spliced transcripts, allelic gene variants and single nucleotide polymorphisms.

– Higher dynamic range of expression levels over which transcripts can be detected (>8000 fold)

– Higher reproducibility

– Lower technical variation.

Question 26

Differential expression analysis

Answer 26

Method by which read count data are analysed to discover quantitative changes in expression levels between experimental groups.

Question 27

Heat map

Answer 27

A common method of visualizing gene expression data, where data are displayed in a grid where each row represents a gene and each column represents a sample. The colour and intensity of the boxes is used to represent changes in gene expression relative to a reference condition.

Question 28

The Gene Ontology (GO) project

Answer 28

Provides a framework and set of concepts for describing the functions of gene products from all organisms.

Question 29

KEGG pathway

Answer 29

A collection of manually drawn pathway maps representing our knowledge of the molecular interactions and relation networks for processes such as metabolism, disease (immune and endocrine systems), cellular processes (such as growth), etc.

Question 30

proteomics applications

Answer 30

Recovery and analysis of proteins from the marine environment (e.g. understanding mechanisms that control the degradation of organic matter)

Question 31

western blotting method

Answer 31

Prepare protein mixture- protein isolation

• Run mixture on a gel
• Stick proteins to a surface
• ‘Probe’ the surface with antibody raised against your protein of interest
• Couple antibody to some form of detectable signal (mainly fluorescent and chemiluminescent)

Question 32

metabolome

Answer 32

set of naturally occurring small molecules (metabolites)

Question 33

metabolomics applications

Answer 33

• Understanding mechanisms by which animals respond to environmental stimuli (e.g. pollution)
• Identification of molecules that can act as biomarkers to identify ecosystem health.
• Areas of application of environmental metabolomics: aquatic toxicology, terrestrial toxicology, fish diseases, aquaculture, environmental monitoring and ecological risk assessment.

Question 34

Elucidating gene function: Gene knock down via RNAi (dsRNA)

Answer 34

Rather than inhibiting translation, RNAi works by degrading the targeted message.

Question 35

Elucidating gene function:
Gene knockout (and editing) via CRISPR

Answer 35

• Cas9 recognizes a DNA sequence Protospacer Adjacent Motif (PAM, 2-6 bp in length) sequence to the 3’ of the target sequence.
• After binding the DNA, Cas9 will cause a double stranded break at the target site.
• By providing a donor DNA, a transgenic DNA can be created.
• In the absence of a donor DNA, the double strand break will be repaired by the host cell, resulting in an insertion or deletion, thus potentially disrupting the open reading frame of a gene.