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TRANSCRIPT
DNA Sequencing
Happiness Kumburu BSU-‐workshop Nov, 2016
OUT LINE
• History of DNA sequencing • Purpose of DNA sequencing • DNA Sequencing Methods • Advantages and Disadvantages • References
DNA SEQUENCING DNA sequencing-the process of determining the precise order of nucleotides within a DNA molecule.
1950 1960
Discovery of DNA structure by Watson and Crick (1953)
1970 1980
Development of Sanger Sequencing by Frederick Sanger (1977)
Polymerase Chain Reaction (PCR) Invented by Kary Mullis (1983)
Human Genome Project Initiated Development of Pyrosequencing by Pål Nyrén
1990 2000
454 Life Sciences Corp. is founded
Sequence of Human Genome Completed (2003)
454 Sequencing™ was used to sequence the following genomes (along with many others): - Barley (plant) - Neanderthal - Helicobacter pylori (H.pylori - bacteria that cause gastric ulcers) - HIV mutant strains
History of Genome Sequencing
PURPOSE OF SEQUENCING
• Containing code or instructions for building an org.
• Ensures that org
functions correctly
• Knowledge of DNA sequences has been applied in fields such as:
• diagnostic, • biotechnology, • forensic biology and • biological systematic
DNA SEQUENCING METHODS
• 1st Generation Sequencing Methods • 2nd Generation Sequencing Methods –NEXT –GENERATION
sequencing methods ( includes Sanger method which next generation sequencing is based on)
• 3rd Generation sequencing methods
Sanger sequencing method (1st generation)
Based on modification of dNTP
DNA sample divided into 4 separate reactions to normal (NTP) and ONE of ddNTPs are added.
Visualizing the products in gel electrophoresis
Next generation sequencing (2nd generation)
• Since 2005 new methods are emerging that
challenge the supremacy of the dideoxy method. • High-throughput • High coverage • High accuracy
• A few examples of specific instruments that employ massively parallel strategies include:-
• Roche 454 sequencing : The first of the
massively parallel method by 454 Life Sciences
• Illumina (Solexa) sequencing : Illumina (Solexa) sequencing .
– ABI’s SOLiD sequencing: Applied Biosystems
Supported Oligonucleotide Ligation and Detection system (SOLiD)
– Pacific Biosciences SMRT DNA sequencing : very long reads, ultra-fast cycle times, and the flexibility to cost-effectively perform small or large projects.
Illumina (Solexa) Sequencing Technology
Modified dNTPs containing a terminator which blocks further polymerization .
Illumina (Solexa) sequencing
-‐making DNA library (~300bp fragments) -‐ligaKon of adapters A and B to the fragments
-‐ “””””barcode sequences are added to each sample so they can be differenKated during the data analysis.
Illumina (Solexa) sequencing
Bridge amplificaKon: iniKaKon
GeneCore
On the surface: complementary oligos
Illumina (Solexa) sequencing
EMBL Gene Core
What are the differences?
• CE-based Sanger Sequencing
– Library preparation more involved—each sample must contain a single template,
– Requiring DNA purification from single organism
– Complete within days to weeks, depending upon the size of the genome being sequenced
• Next-Generation Sequencing
– Streamlined library prep—sample can consist of a
– No clonal purification of DNA. – Completed within hours, regardless of genome size
Third generation sequencing • Based on modification of the second generation
sequencing • 2 main characteristics
– No PCR before sequencing – Signal captured in real time
• Examples – Single molecule real time (SMRT) by Pacific
Biosciences – Nanopore sequencing
Whole Genome Sequencing On Miseq At KCM/KCRI-biotechnology Laboratory
• .
WGS-Is a laboratory process that determines the complete DNA sequence of an organism's genome at a single time
WARDS Consent forms CRFs Specimens Specimen forms
LABORATORY
Specimens
Specimen forms
Isolates
Bio repository
DMU
Consent forms
CRFs
Analysis
Analysis Results
WGS
Work flow chart
NGS ON
MISEQ
ConvenKonal microbiology
analysis
Blood samples sputum Wound/pus swabs from infected
wounds
Stool samples Wound/pus swabs from infected wounds
Culture, Identification, DST, DNA extraction ,Library prep and Sequencing
Our analysis
• Raw sequence data – Spp confirmation KmerFinder , MLST (version 1.7), ResFinder (version 2.1) and VirulenceFinder (version 1.02 available from CGE
(http://cge.cbs.dtu.dk/services/all.php). • Further phylogenetic analysis • SNP analysis • Temporal Phylogenetic analysis-divergence times,
mutation rates, population structure
ADVANTAGES OF DNASEQUENCING
• Molecular medicine
• Bioarchaeology, anthropology, evolution, and human migration
• DNA forensics (identification) • Agriculture, livestock breeding, and
bioprocessing
DISADVANTAGES OF DNA SEQUENCING
• Too expensive • Religious beliefs • Invasion of individual privacy
REFERENCES
• DNA Sequencing Protocols. Editors: Graham, Colin A, Hill, Alison J.M. (Eds.)
• The sequence of sequencers: The history of sequencing DNA. Heather JM, Chain B.
• http://www.illumina.com/techniques/sequencing/dna-sequencing.html • High-throughput DNA sequencing – concepts and limitations. Martin
Kircher, Janet Kels • Comparison of Next-Generation Sequencing Systems • Lin Liu et al ,2012.
THANK YOU
h^ps://www.youtube.com/watch?v=womKfikWlxM