Lecture 29: Analysing Microbial Genomes by NGS

Question 1

What is next gen sequencing?

Answer 1

1, Complex and advanced tech

2. Relies on:
   a. modern, high power computing
   b. Extensive engineering
   c. Nanoscale engineering
   d. Modern, advanced materials

Question 2

What is illumina sequencing?

Answer 2

1. “Sequencing b synthesis”
2. Process:
   a. DNA amplified on a slide
   b. Fluorescent terminator base anneals
   c. Laser removes fluorescent terminator
   d. Fluorescent label read
   e. Process repeats

Question 3

What is PAC-BIO sequencing

Answer 3

1. Detection of fluorescence from labelled
   nucleotides during DNA synthesis
2. Occurs in arrays of small wells

Question 4

What would you use PacBio/MinIon for?

Answer 4

1. Long reads
2. Generating gold-standard reference
   genomes
3. Reconstruct intact genes and gene
   clusters
4. E.g., ANTIBIOTIC DISCOVERY

Question 5

What would you use Illumina for?

Answer 5

1. Fast, high throughput, low cost/bp
2. Allows metagenomic analysis of complex
   environments
3. Can compare organisms cheaply

Question 6

What are transcriptional reporter fusions?

Answer 6

1. If you want to understand how a gene is
   regulated.
   2, Take something that glows or has
   specific output (LacZ blue/white, GFP)
2. Compare control with sample of KO TF

Question 7

What is RNA seq?

Answer 7

1. Microarrays, qRT-PCR rely on hybridisation

Question 8

What are the pros and cons of RNA seq

Answer 8

\+ All transcription is studied in an 
   unbiased manner
\+ more precise - higher resolution
\+ No interference from non-specific 
   hybridization 
\+ Allows discovery of novel genetic 
   features, delineation of operons and 
   untranslated regions
\+ greater dynamic range, more   
   discrimitory and sensitive

Question 9

What is chromatin immunoprecipitation (ChIP) -seq?

Answer 9

1. Identification of all the binding sites for a

DNA binding proteins in a genome