This presensation consist of introduction and little bit introduction of DNA sequencing and its types with graphical explanation of there each types

1. Sequencing Techniques
Faisalabad Institute of Research Science and Technology
(FIRST)
Presented by ::
Taimoor Akhter
Bs.c (Hons) biotechnology

2. Outline
• Definition
• Introduction
• History
• Early methods
• New techniques
• Application
• Defects in sequencing techniques

3. Deoxyribonucleic Acid
• Deoxyribonucleic acid (DNA) is a nucleic acid that functions include
• Storage of genetic information
• Self-duplication & inheritance
• Expression of the genetic message
• DNA’s major function is to code for proteins. Information is encoded in
the order of the nitrogenous bases.
Adenosine Cytosine Guanine Thymine

4. Watson & Crick Model of DNA
• DNA is composed of 2 chains of nucleotides
that form a double helix shape.
• The two strands are antiparallel.
• The backbone of the DNA molecule is composed of
alternating phosphate groups and sugars.
• The complimentary nitrogenous bases form
hydrogen bonds between the strands.
• A is complimentary to T and G is complimentary
to C.

5. What is sequencing
• DNA sequencing is the process of determining the sequence of
nucleotides within a DNA molecule.
• Every organism’s DNA consists of a unique sequence of
nucleotides.
• Determining the sequence can help scientists compare DNA
between organisms, which can help show how the organisms
are related.
• Used to determine the sequence of individual genes, larger genetic
regions, full chromosomes or entire genomes.
• The resulting sequences may be used by researchers in molecular
biology or genetics to further scientific progress.

6. PURPOSE
• Understanding “code of cell life”
• Detecting mutations
• Typing microorganisms
• Identifying human halotypes
• Designating polymorphisms

7. History of Sequencing
• 1972 – Earliest nucleotide sequencing – RNA sequencing of Bacteriophage
MS2 by WALTER FIESERR
• Early sequencing was performed with tRNA through a technique developed
by Richard Holley, who published the first structure of a tRNA in 1964.
• 1977 - DNA sequencing FREDRICK SANGER by Chain termination method
• Chemical degradation method by ALLAN MAXAM and WALTER GILBERT
• 1977 - First DNA genome t be sequenced of Bacteriophage ΦX174
• 1986 - LOREY and SMITH gave Semiautomated sequencing
• 1987 – Applied biosystems marketed Fully automated sequencing
machines

8. DNA SEQUENCING METHODS
1) Maxam and Gilbert chemical degradation method
2) Chain termination or Dideoxy method
• Fredrick Sanger
3) Genome sequencing method
• Shotgun sequencing
• Clone contig approach
4) 2nd generation sequencing methods
• Pyrosequencing
• Nanopore sequencing
• Illumina sequencing
• Solid sequencing

9. Maxam and Gilbert chemical degradation method
• A. M. Maxam and W.Gilbert-1977
• Chemical Sequencing
• Treatment of DNA with certain Chemicals
(formic acid, dimethyl sulfate, hydrazine,
hydrazine, piperidine).
• DNA cuts into Fragments
• Monitoring of sequences

11. Advantages
• Improved diagnosis of disease
• Bio pesticides
• Identifying crime suspects
Disadvantages
• Whole genome cannot be sequenced at once
• Very slow and time consuming

12. SANGER METHOD
Most common approach used for DNA
sequencing .
• Invented by Frederick Sanger - 1977
• Nobel prize - 1980
• Also termed as Chain Termination or
Dideoxy method

13. Chain termination method of DNA
sequencing.
It involves following components:
a) Primer
b) DNA template
c) DNA polymerase
d) dNTPs(A,T,G,C)
e) ddNTPs

14. Principle
ssDNA
Enzymatic synthesis of complementary polynucleotide chains
Termination at specific nucleotide positions
Separate by Gel Electrophoresis
• Read DNA Sequence

15. steps
1. Denaturation
2. Primer attachment and extension of bases
3. Termination
4. Poly acrylamide gel electrophoresis

17. Other types of sequencing
Pyrosequencing
• Pyrosequencing is the second important type of DNA
sequencing methodology in use today.
• The method amplifies DNA inside water droplets in an oil
solution (emulsion PCR), with each droplet containing a single
DNA template attached to a single primer-coated bead that
then forms a clonal colony.
• The sequencing machine contains many picoliter-volume wells
each containing a single bead and sequencing enzymes.
• Pyrosequencing uses luciferase to generate light for detection
of the individual nucleotides added to the nascent DNA, and
the combined data are used to generate sequence reads.

19. Advantages
• Accurate
• Parallel processing
• Easily automated
• Eliminates the need for labeled primers and nucleotides
• No need for gel electrophoresis
Disadvantages
• Only for smaller sequences
• Expensive
• Laborous

20. Shotgun sequencing
• Shotgun sequencing, also known as shotgun cloning, is a method
used for sequencing long DNA strands or the whole genome.
• •In shotgun sequencing, DNA is broken up randomly into numerous
small segments and overlapping regions are identified between all
the individual sequences that are generated.
• • Multiple overlapping reads for the target DNA are obtained by
performing several rounds of this fragmentation and sequencing.
• •Computer programs then use the overlapping ends of different
reads to assemble them into a continuous sequence.
• •The shotgun approach was first used successfully with the bacterium
Haemophilus influenzae.

22. Advantages
• Faster because the mapping process was eliminated
• Uses less DNA than other methods
• Less expensive than approaches requiring a map
Disadvantages
• Requires computer processing power beyond what an ordinary
laboratory would possess
• Can introduce errors in the assembly process
• Requires a reference genome
• May not be able to assemble repetitive sequences

23. Nanopore sequencing
• The fourth-generation DNA sequencing technology.
• Studies the interaction between DNA and protein, as well as between
protein and protein
• Nanopore sequencing uses electrophoresis to transport an unknown
sample through an orifice of 10−9 meters in diameter.

24. Conti…….
Principle
The detection principle is based on monitoring the ionic current
passing through the nanopore as a voltage is applied across the
membrane. When the nanopore is of molecular dimensions, passage of
molecules (e.g., DNA) cause interruptions of the current level, leading
to a signal.

25. TYPES OF NANOPORES
• Biological nanopore
• Solid-state nanopore
• Hybrid nanopore

26. Advantages
• minimal sample preparation
• no requirement for ploymerase and ligase
• potential of very long read-lengths ( > 10,000 – 50,000 nt )
• it might well achieve the $1,000 per mammalian genome goal
• the instrument is inexpensive

27. Illumina sequencing
Illumina dye sequencing is a technique used to determine the series of
base pairs in DNA, also known as DNA sequencing. ...
This sequencing method is based on reversible dye-terminators that
enable the identification of single bases as they are introduced into
DNA strands.

28. Steps in illumina sequencing
• Tagmentation
• Reduced cycle amplification
• Reduced cycle amplification
• Clonal amplification
• Sequence by synthesis
• Data analysis

30. Advantages
• High throughput / cost.
• Suitable for a wide rage of applications most notably whole genome
sequencing.
Disadvantages
• Substitution error rates (recently improved).
• Lagging strand dephasing causes sequence quality deterioration
towards the end of read.

31. Application of sequencing
• DNA sequencing may be used to determine the sequence of
individual genes, larger genetic regions (i.e. clusters of genes
or operons), full chromosomes, or entire genomes of any organism.
• DNA sequencing is also the most efficient way to indirectly
sequence RNA or proteins (via their open reading frames).
• In fact, DNA sequencing has become a key technology in many areas
of biology and other sciences such as medicine, forensics,
and anthropology.

32. Conti…
• Molecular biology
• Evolutionary biology
• Metagenomics
• Medicine
• Forensics