Lecture 29: Analysing Microbial Genomes by NGS
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What is next gen sequencing?
1, Complex and advanced tech • Relies on: a. modern, high power computing b. Extensive engineering c. Nanoscale engineering d. Modern, advanced materials
What is illumina sequencing?
• “Sequencing b synthesis” • Process: a. DNA amplified on a slide b. Fluorescent terminator base anneals c. Laser removes fluorescent terminator d. Fluorescent label read e. Process repeats
What is PAC-BIO sequencing
• Detection of fluorescence from labelled nucleotides during DNA synthesis • Occurs in arrays of small wells
What would you use PacBio/MinIon for?
• Long reads • Generating gold-standard reference genomes • Reconstruct intact genes and gene clusters • E.g., ANTIBIOTIC DISCOVERY
What would you use Illumina for?
• Fast, high throughput, low cost/bp • Allows metagenomic analysis of complex environments • Can compare organisms cheaply
What are transcriptional reporter fusions?
• If you want to understand how a gene is regulated. 2, Take something that glows or has specific output (LacZ blue/white, GFP) • Compare control with sample of KO TF
What is RNA seq?
• Microarrays, qRT-PCR rely on hybridisation
What are the pros and cons of RNA seq
\+ 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
What is chromatin immunoprecipitation (ChIP) -seq?
• Identification of all the binding sites for a DNA binding proteins in a genome