Environmental genomics and applications

Question 1

What is the basic principle of DNA sequencing techniques?

Answer 1

Capillary-based Sanger sequencing
Next generation sequencing:
- Short read (Illumina)
- Long read (PacBio)

Question 2

What is capillary-based Sanger sequencing?

Answer 2

• Single DNA fragment
• Fast turnaround with ~700bp
• Early genome sequencing
• laborious + time + money
• 16s rRNA gene

Question 3

What is a shotgun method?

Answer 3

Shotgun sequencing involves randomly breaking up DNA sequences into lots of small pieces and then reassembling the sequence by looking for regions of overlap.

Question 4

What is Illumina sequencing in a flow cell?

Answer 4

1) attach DNA fragments to flow cell.
2) Strand synthesis and cluster formation.
3) Laser excitation - image bases in each cluster.
- barcode samples before sequencing

Question 5

What is omics?

Answer 5

Sequences based:
DNA (Function potential )
 - A genome is a metagenome (environmental genomics)
Messenger RNA (Function )
 - Transcriptome is metatranscriptome.
Chromatography/ Mass spectrometry-based:
Protein (Function)
- Proteome is metaproteome.
Metabolite (product)
- Metabolome

Question 6

What is genomics?

Answer 6

• It is DNA (functional potential), collection of genes
• Bacteria DNA found on singular circular chromosome.
  some have multiple and linear chromosomes, while other genes are found on plasmids.
• Genome can include viral DNA (bacteriophage).
• A large de Brujin graph constructed from mixed reads of multhiple species.

Question 7

What is metagenomics (environmental genomics) ?

Answer 7

Sequence DNA in a sample from mixtures of uncultivated organisms. This helps us to understand metabolic potential (insight in to the lifestyle of uncultivated microorganisms) and potential guide for cultivation efforts.

Question 8

Why there are so many uncultivated?

Answer 8

Many are recalcitrance to conventional cultivation techniques that target weeds (r-strategists) and exclude slower growing taxa ( K- strategists) and metabolically dependent prokaryotes (auxotrophs).

Question 9

What are the metagenomic sequencing approaches?

Answer 9

Sanger sequencing

Next-generation sequencing (common approach eg. Illumina)

Question 10

What is the computationally challenging method?

Answer 10

• easiest for small simple genomes, eg. viruses, bacteria and archaea.
• recovering more complex microbial eukaryotes genomes

Question 11

What computationally challenging method?

Answer 11

• better understand on genome lengths and coding DNA content.
• Expanding the tree of life with environmental genomics
• Predictions of microbial interactions

Question 12

What is transcriptomics (metatranscriptomics)?

Answer 12

• Measurement of gene expression via RNA sequencing
• most DNA is ribosomal RNA (rRNA) and a smaller fraction is messenger RNA (mRNA), which is used as a proxy for protein expression.

Question 13

What is proteomics (metaproteomics)?

Answer 13

• Mass spectroscopy
• determine protein expression and metabolic activity.
• top-down proteomics: measure single whole proteins
• Bottom-up (shotgun) proteomics: Measure all proteins and the first proteins are digested into peptides

Question 14

What is metabolomics?

Answer 14

• Mass spectroscopy
• Metabolites: substrates and products of metabolism
• Measurements: liquid chromatography coupled with mass spectrometry (LC-MS), Gas chromatography coupled with mass spectrometry (GC-MS), Nuclear magnetic resonance (NMR) spectrometry.