N G S TECHNOLOGY
NEXT GENERATION SEQUENCING
DENATURATION/HYBRIDIZATION
Denaturation
Hybridization / Annealing
Separating DNA into single
Forming double-stranded DNA
strands
NU C L E I C ACID THERMODYNAMICS SINGLE 2 DU PL EX
○ Hybridization is the process of establishing a non-
covalent, sequence-specific interaction between two or more
complementary strands of nucleic acids (i.e. DNA/RNA) into
a single hybrid, which in the case of two strands is referred
to as a duplex
○ Annealing, in genetics, means for D NA or R NA to pair by
hydrogen bonds to a complementary sequence, forming a
double-stranded polynucleotide. The term is often used to
describe the binding of a D NA probe, or the binding of a
primer to a D NA strand during a polymerase chain reaction
(PCR). The term is also often used to describe the
reformation (renaturation) of complementary strands that
were separated by heat (thermally denatured)
NU C L E I C ACID THERMODYNAMICS DUPLEX 2 S I N G L E
○ D N A denaturation, also called D N A melting, is the
process by which double-stranded deoxyribonucleic acid
(DNA) unwinds and separates into single-stranded strands
through the breaking of hydrogen bonding between the
bases. Both terms are used to refer to the process as it
occurs when a mixture is heated, although "denaturation"
can also refer to the separation of D NA strands induced by
chemicals
○ For multiple copies of D NA molecules, the melting
temperature (Tm) is defined as the temperature at which
half of the D NA strands are in the double-helical state and
half are in the random coil or single-stranded state.
PO L Y M E R A S E C H A I N R E A C T I O N (PCR)
○ The polymerase chain reaction (PCR) is a
technique in molecular biology to amplify a single
or a few copies of a piece of D NA across several
orders of magnitude, generating thousands to
millions of copies of a particular D NA sequence.
○ The method relies on thermal cycling, consisting
of cycles of repeated heating and cooling of the
reaction for D NA melting and enzymatic
replication of the D NA
PO L Y M E R A S E C H A I N R E A C T I O N (PCR)
(1) Denaturing at 94–96 °C.
(2) Annealing at ~65 °C
(3)Elongation at 72 °C. At this step the DNA
polymerase synthesizes a new DNA strand
complementary to the DNA template strand
by adding bases that are complementary to
the template in 5' to 3' direction, condensing
the 5'-phosphate group of the bases with the
3'-hydroxyl group at the end of the nascent
(extending) DNA strand.
Exponential growing
Four cycles are shown here. The blue lines
represent the DNA template to which primers
(red arrows) anneal. These are extended by
the DNA polymerase (light green circles), to
give shorter DNA products (green lines),
which themselves are used as templates as
PCR progresses.
PO L Y M E R A S E C H A I N R E A C T I O N (PCR)
VIDEO
○ https://www.dnalc.org/resources/animations/pcr.h
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DNA L I B R A R Y P R E P A R A T I O N
○ Most sequencing approaches use an in vitro
cloning step to amplify individual D NA molecules
○ that is because their molecular detection methods
are not sensitive enough for single molecule
sequencing
1. DNA Library preparation
Preparation of the DNA library consists of
a few simple steps:
• Genomic DNA is fractionated into smaller
fragments (300-500 base pairs) that are
subsequently phosphorylated (addition of a
phosphate (PO4) group; phosphorylation
activates or deactivates many protein
enzymes).
• Short Adaptors (A and B) are then ligated onto the ends of the fragments. These
adaptors provide priming sequences for both amplification and sequencing of the
sample-library fragments. Adaptor B contains a 5'-biotin tag that enables immobilization of
the library onto streptavidin coated beads (in fact, Biotin binds very tightly to the tetrameric
protein avidin (also streptavidin) creating one of the strongest known protein-ligand
interactions, approaching the covalent bond in strength; biotin-binding is resistant to
extremes of pH, temperature, organic solvents, denaturants, detergents and proteolytic
enzymes).
1. DNA Library preparation
dsDNA fragments
P Bio
A P B
Ligation
Bio
• Short Adaptors (A and B) are
then ligated onto the ends of the Fill in
fragments. These adaptors provide Bio
priming sequences for both
amplification and sequencing
Bio
Bio
Capture on SA-Beads & Wash
Alkaline Elution
A B
2. CLONAL AMPLIFICATION DURING EMPCR
Emulsion-based clonal amplification
Anneal sstDNA
Emulsify beads and Break
(single strand Clonal ampli cation
PCR reagents in microreactors,
DNA) to an excess occurs inside
water-in-oil enrich for DNA-
of DNA Capture microreactors
microreactors positive beads
Beads
DNA Library Preparation emPCR Sequencing
4.5 h 8h 7.5 h
and 10.5
h
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3. S E Q U E N C I N G - B EAD LOADING ON PICO TITER PLATES
Depositing DNA beads into the PicoTiterPlate device
▪ Well diameter: average of 44 µ
▪ 400,000 reads obtained in paralle
▪ A single clonally ampli ed
sstDNA bead is deposite
per wel
AmplifiedsstDNA library beads Quality ltered bases
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NGS TECHNOLOGY SEQUENCING-BY-SYNTHESIS
Repeated dNTP G T C A
ow sequence:
Process continues until user-
de ned numbe
of nucleotide ow cycles are
completed.
A A T C G G C A T G C T A A A A G T C C
Anneal
T T A G C C G T A C G A T T T T C A G G Primer
Nucleotide Pairs
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The sequencing instrument consists of the
following major subsystems:
A) a fluidic assembly
B) a flow chamber that includes the well-
containing fibre-optic slide
C) a CCD camera-based imaging assembly
D) and, a computer that provides the
necessary user interface and instrument
control.
