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