DNA Sequencing Techniques & Technologies PDF

3-3: Genomics Lecture Overview: • DNA sequencing technologies and how they have revolutionized microbiology. “omics” technologies: genomics, metagenomics, transcriptomics & proteomics. • Sections: 10.1, 10.2, 10.7-10.9 DNA sequencing – First complete genome Craig Venter has been a major name in DN...

3-3: Genomics Lecture Overview: • DNA sequencing technologies and how they have revolutionized microbiology. “omics” technologies: genomics, metagenomics, transcriptomics & proteomics. • Sections: 10.1, 10.2, 10.7-10.9 DNA sequencing – First complete genome Craig Venter has been a major name in DNA sequencing. Used “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 First genome of a free-living organism – bacterium H. influenzae (pathogen, causes meningitis, pneumonia…but doesn’t cause the flu) Published in Science in 1995 DNA sequencing – a revolution As an example, more than 300,000 Salmonella genomes have been fully sequenced (number out of date) & this data is deposited in sequence databases online. DNA sequencing – right here in Alberta DNA sequencing – now very accessible It’s now $90 to sequence a complete genome (including some annotation/data analysis!!!) Done in 1-2 days Wowzers!! (complete genomes no longer get you a paper in the top journal “Science”) DNA sequencing - Sanger o Developed by Fredrick Sanger in 1970s – Nobel prize (one of his two!) o 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 o Special “ddNTPs” each labelled a different way (different fluorophores) o Build DNAs of different lengths, each terminated with a labelled ddNTP o Determine sequence based on identity of terminating residues (e.g. – 26 nt sequence terminated with a “T”, 27 nt sequence terminated with a “G”, etc.) Sanger sequencing – still used today for smaller sequencing projects DNA sequencing - Sanger Strand to be sequenced! 3’OH missing in ddNTPs! DNA polymerase 5’ P P P T OH-3’ Short DNA primer Template strand Modified from image at www.goldbio.com DNA sequencing: “Next generation” – massively parallel sequencing o Different variations - Illumina sequencing popular o Chips generate millions of “clusters”, each represents (many copies of) a different DNA molecule being sequenced Reversibly terminated dNTPs used: o Insert one labelled residue, take an image (A, C, G, T each a different colour of fluorophore) o Unblock the 3’ end so you can add another residue. Repeat. o Each round, you get an image of what residue is at each cluster for that position (1st nt, 2nd, 3rd…) Illumina image – each dot a different cluster. Next round a new image of the same clusters will be taken to reveal the sequence of the next nt DNA sequencing: annotation o DNA sequencing has limited utility without annotation – identifying the genes, their putative functions, etc. o Largely done automatically (computers)…useful but many mistakes, things missed, incorrect assumptions o Identify genes based on homology to known genes – many “unknown function” or “putative XXXX” o Annotation & analysis lag behind sequencing Textbook Fig 10.6 An example of how computers might identify an ORF (open reading frame – protein-coding sequence) Genome sequencing – what can it tell us? Asgard Archeas What we know about these all from genome Sequencing ... comes genes , metabolism , virulence .... etc. . What genes are present/absent & the sequences of each gene o Metabolic capabilities of an organism things o Virulence genes, antibiotic resistance genes, etc o Unusual mutations that account for unusual phenotypes o Discover new genes that might be of medical/industrial interest o etc… I can * a even tell as bout a we lot already . know ↳ S peci fic resistance Provides DNA blueprint required for many studies/analyses o Genetics approaches (e.g. making mutations to genes) Lygene o Transcriptomics, qPCR, etc – studies of RNA expression o Proteomics – studies of proteins o Genome-wide mutagenesis studies (looking at the effects of many different mutations in parallel) ↳Needs DNA blueprints for .... deletions Antibiotic Genome sequencing – what can it tell us? Functional & metabolic predictions for a Vampirovibrio chlorellavorus strain based on its genome sequence. Metagenomics o Metagenomics is the study of the complete genetic content of an environmental sample o Massive sequencing of DNA purified from environmental samples can provide genomic information (sometimes even complete genomes) for organisms that cannot be cultured in the lab o Can tell us about the composition of microbial communities & how that composition changes o Can also look at gene level. E.g. – how does the frequency of antibiotic resistance genes compare in the microbiomes of animals from different farms? Textbook - Fig 10.17 Transcriptomics: RNA-seq o RNA can be converted to DNA using a process called reverse transcription, which can then be sequenced via next-generation methods o Can get a complete picture of the relative abundance of all of the transcripts in the cell under a given set of conditions o One way this is used is comparing expression under different conditions. E.g. – which genes does a pathogen specifically express during infection? RNA-seq identifies S. aureus genes whose expression goes up (green) or down (red/orange) during infection compared to when grow in the lab Ibberson & Whiteley, mBio, 2019 Proteomics o Another important “omics” technology is proteomics. o Often relies on knowing the genomic DNA sequence, but doesn’t use DNA sequencing. o Instead, uses mass spectrometry to identify proteins/protein levels o 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 Textbook - Fig 10.23