Topic 5-L3 - Genomics

Question 1

Craig Venter has been a major name in DNA sequencing. Used

Answer 1

“Shotgun sequencing” – sequence random bits of DNA, let computers figure out how it all fits together. Faster/more efficient than more structured approach used originally

Question 2

DNA sequencing - Sanger

Answer 2

Based on DNA polymerase building a complementary strand using: (i) mostly normal dNTPs and (ii) rare special dNTPs that lack a 3’OH and therefore cannot be elongated further

- Special “ddNTPs” each labelled a
different way (different fluorophores) 

• Build DNAs of different lengths, each
  terminated with a labelled ddNTP

Question 3

DNA sequencing: “Next generation” – massively parallel sequencing

Answer 3

Chips generate millions of “clusters”, each represents (many copies of) a different DNA molecule being sequenced

Question 4

Reversibly terminated dNTPs used:

Answer 4

• Insert one labelled residue, take an image (A, C, G, T each a different colour of fluorophore)
• Unblock the 3’ end so you can add
  another residue. Repeat.
• Each round, you get an image of what residue is at each cluster for that position (1st, 2nd, 3rd…)

Question 5

DNA sequencing has limited utility without annotation –

Answer 5

identifying the genes, their putative functions, etc.

Question 6

Annotations largely done via

Answer 6

Computers, useful but mistakes are made

Question 7

What genes are present, what genes are absent & the sequences of individual genes

Answer 7

• Metabolic capabilities of an organism
• Virulence genes, antibiotic resistance genes, etc
• Unusual mutations that account for unusual phenotypes
• Discover new genes that might be of medical/industrial interest

Question 8

Provides DNA blueprint required for many types of studies/analyses

Answer 8

• Genetics approaches (e.g. making mutations to genes)
• Transcriptomics, qPCR, etc – studies of RNA expression
• Proteomics – studies of proteins
• Genome-wide mutagenesis studies (looking at the effects of many different mutations in parallel)

Question 9

Metagenomics is the study of the

Answer 9

complete genetic content of an

environmental sample

Question 10

How are metagenomics made

Answer 10

Massive sequencing of samples of DNA purified from environmental samples can provide genomic information (sometimes even complete genomes) for organisms that cannot be cultured in the lab

Question 11

Metagenomics can tell us about

Answer 11

Microbial communities and gene level

Question 12

gene level

Answer 12

E.g. – how does the frequency of antibiotic resistance genes compare in the microbiomes of animals from different
farms?

Question 13

Transcriptomics: RNA-seq

Answer 13

RNA can be converted to DNA using a process called reverse transcription, which can then be sequenced via next-generation methods

Question 14

Proteomics

Answer 14

• Often relies on knowing the genomic DNA sequence, but doesn’t use DNA sequencing.
• Instead, uses mass spectrometry to identify proteins/protein levels
• Like RNA-seq, can tell you what proteins are present under which
  conditions. Can also be used in many other creative ways. E.g. which
  proteins interact with a protein of interest