DNA Sequencing. Happiness Kumburu BSU- workshop Nov, 2016

Transcription

1 DNA Sequencing Happiness Kumburu BSU- workshop Nov, 2016

2 OUT LINE History of DNA sequencing Purpose of DNA sequencing DNA Sequencing Methods Advantages and Disadvantages References

3 DNA SEQUENCING DNA sequencing-the process of determining the precise order of nucleotides within a DNA molecule.

4 History of Genome Sequencing Discovery of DNA structure by Watson and Crick (1953) Development of Sanger Sequencing by Frederick Sanger (1977) Polymerase Chain Reaction (PCR) Invented by Kary Mullis (1983) Human Genome Project Initiated 454 Life Sciences Corp. is founded Sequence of Human Genome Completed (2003) Development of Pyrosequencing by Pål Nyrén 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

5 PURPOSE OF SEQUENCING Containing code or instructions for building an org. Ensures that org functions correctly

6 Knowledge of DNA sequences has been applied in fields such as: diagnostic, biotechnology, forensic biology and biological systematic

7 DNA SEQUENCING METHODS 1 st Generation Sequencing Methods 2 nd Generation Sequencing Methods NEXT GENERATION sequencing methods ( includes Sanger method which next generation sequencing is based on) 3 rd Generation sequencing methods

8 Sanger sequencing method (1 st generation) Based on modification of dntp

9 DNA sample divided into 4 separate reactions to normal (NTP) and ONE of ddntps are added.

10 Visualizing the products in gel electrophoresis

11 Next generation sequencing (2 nd generation) Since 2005 new methods are emerging that challenge the supremacy of the dideoxy method. High-throughput High coverage High accuracy

12 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.

13 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.

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15 Illumina (Solexa) Sequencing Technology Modified dntps containing a terminator which blocks further polymerization.

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17 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.

18 Illumina (Solexa) sequencing Bridge amplificakon: inikakon On the surface: complementary oligos GeneCore

19 Illumina (Solexa) sequencing EMBL Gene Core

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21 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

22 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

23 Whole Genome Sequencing On Miseq At KCM/KCRIbiotechnology Laboratory. WGS-Is a laboratory process that determines the complete DNA sequence of an organism's genome at a single time

24 WARDS Consent forms CRFs Specimens Specimen forms Work flow chart LABORATORY Specimens Results DMU Consent forms Analysis Specimen forms ConvenKonal microbiology CRFs Isolates analysis NGS ON MISEQ WGS Analysis Bio repository

25 Blood samples Stool samples sputum Wound/pus swabs from infected wounds Wound/pus swabs from infected wounds

26 Culture, Identification, DST, DNA extraction,library prep and Sequencing

27 Our analysis Raw sequence data Spp confirmation KmerFinder, MLST (version 1.7), ResFinder (version 2.1) and VirulenceFinder (version available from CGE ( Further phylogenetic analysis SNP analysis Temporal Phylogenetic analysis-divergence times, mutation rates, population structure

28 ADVANTAGES OF DNASEQUENCING Molecular medicine Bioarchaeology, anthropology, evolution, and human migration DNA forensics (identification) Agriculture, livestock breeding, and bioprocessing

29 DISADVANTAGES OF DNA SEQUENCING Too expensive Religious beliefs Invasion of individual privacy

30 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. High-throughput DNA sequencing concepts and limitations. Martin Kircher, Janet Kels Comparison of Next-Generation Sequencing Systems Lin Liu et al,2012.

31 THANK YOU

32 h^ps:// v=womkfikwlxm