Next generation sequencing techniques allow DNA to be sequenced much more quickly and cheaply than previous Sanger sequencing. The document describes three main next generation sequencing methods: Illumina sequencing uses reversible dye-terminator nucleotides and DNA polymerase to sequence DNA clusters on a flowcell; Roche 454 sequencing is based on pyrosequencing and detects pyrophosphate release during nucleotide incorporation to sequence DNA; SOLiD sequencing uses DNA ligase and emulsion PCR to immobilize DNA on beads for sequencing. These new techniques have revolutionized genomics research by increasing sequencing speed and reducing costs.

1. Next generation sequencing
By
Neelma Nayab
MPHIL 2ND
SEMESTER
BT320192021
Department of Biotechnology and Genetic Engineering
Kohat University of Science and Technology, Kohat 26000
Khyber Pakhtunkhwa, Pakistan
June 2020

2. WHAT IS SEQUENCING?
Genome sequencing is a method used to figure out the order of DNA
nucleotides, or bases in a genome-the order of A, C, G, and T that make
up an organism's DNA.
 In Sanger sequencing, the target DNA is copied many times, making
fragments of different lengths. Fluorescent “chain terminator”
nucleotides mark the ends of the fragments and allow the sequence to
be determined.
 Next-generation sequencing techniques are new, large-scale
approaches that increase the speed and reduce the cost of DNA
sequencing.


3. IMPORTANT OF DNA SEQUENCING:
 Better understanding of gene expression. Gene expression
has significance in protein creation etc.
 It is capable detect various diseases and genetic illnesses.
 Personalized medicine and disease discovery is possible.
 Forensics
NEXET GENERATION SEQUENCING:
Next-generation sequencing (NGS), also known as high-
throughput sequencing, is the catch-all term used to describe
a number of different modern sequencing technologies
including:
ILLUMINA (SOLEXA) SEQUENCING
ROCHE 454 SEQUENCING (PYROSEQUENCING)
SOLID SEQUENCING
ION TORRENT: PROTON / PGM SEQUENCING
These recent technologies allow us to sequence DNA and
RNA much more quickly and cheaply than the previously
used Sanger sequencing, and as such have revolutionized
the study of genomics and molecular biology. NGS has
brought high speed not only to genome sequencing and
personal medicine it has also changed the way we do
genome research.
OVERVIEW OF NEXT GENERATION SEQUENCING
PROTOCOL

4. (Fig1)
1 ILLUMINA (SOLEXA) SEQUENCING
Illumina Genome Analyzer is a high-throughput, short-read, massively
parallel sequencing platform. The Illumina Solexa sequencing
technology uses sequencing-by-synthesis on an eight-channel flowcell to
produce more than 10 million reads per channel with read lengths up to
100bp. Individual fragments of a genomic DNA library are amplified on

5. a flowcell via bridge-PCR to generate clusters of identical fragments.
Reversible terminator nucleotides are used in sequencing allowing for
the reading of one base per sequencing cycle per cluster. This platform
enables many applications, including whole genome resequencing,
transcriptome sequencing, gene expression profiling, and epigenomic
sequencing.
How does Illumina DNA sequencing work?
1. The first step in this sequencing technique is to break up the DNA
?into more manageable fragments of around 200 to 600 base pairs?.
2. Short sequences of DNA called adaptors?, are attached to the DNA
fragments.
3. The DNA fragments attached to adaptors are then made single
stranded. This is done by incubating the fragments with sodium
hydroxide.
4. Once prepared, the DNA fragments are washed across the flowcell.
The complementary DNA binds to primers? on the surface of the
flowcell and DNA that doesn’t attach is washed away.
5. The DNA attached to the flowcell is then replicated to form small
clusters of DNA with the same sequence. When sequenced, each cluster
of DNA molecules will emit a signal that is strong enough to be detected
by a camera.
6. Unlabelled nucleotide bases? and DNA polymerase? are then added
to lengthen and join the strands of DNA attached to the flowcell. This
creates ‘bridges’ of double-stranded DNA between the primers on the
flowcell surface.
7. The double-stranded DNA is then broken down into single-
stranded DNA using heat, leaving several million dense clusters of
identical DNA sequences.
8. Primers and fluorescently?-labelled terminators (terminators are a
version of nucleotide base – A, C, G or T - that stop DNA synthesis) are
added to the flowcell.
9. The primer attaches to the DNA being sequenced.

6. 10. The DNA polymerase then binds to the primer and adds the first
fluorescently-labelled terminator to the new DNA strand. Once a base
has been added no more bases can be added to the strand of DNA until
the terminator base is cut from the DNA.
11. Lasers are passed over the flowcell to activate the fluorescent label
on the nucleotide base. This fluorescence is detected by a camera and
recorded on a computer. Each of the terminator bases (A, C, G and T)
give off a different colour.
12. The fluorescently-labelled terminator group is then removed from
the first base and the next fluorescently-labelled terminator base can be
added alongside. And so the process continues until millions of clusters
have been sequenced.
13. The DNA sequence is analysed base-by-base during Illumina
sequencing, making it a highly accurate method. The sequence generated
can then be aligned to a reference sequence, this looks for matches or
changes in the sequenced DNA.(fig 2,3)

7. (Fig 2)

8. (Fig 3)
2 ROCHE 454 SEQUENCING (PYROSEQUENCING)
 A method of DNA sequencing based on
the
“sequencing by synthesis" principle.
 It differs from Sanger sequencing, relying on the
detection of pyrophosphate release (hence the
name) on nucleotide incorporation, rather than
chain termination with dideoxynucleotides.

9.  ssDNA template is hybridized to a sequencing primer
 Incubated with the enzymes DNA
polymerase, ATP sulfurylase,
luciferase and apyrase, and with the
substrates adenosine5´phosphosulfate
(APS) and luciferin.
PYROSEQUENCING CHEMISTRY:

10. PYRO
SEQUENCING
PYROSEQUENCING PROTOCOL: (Fig 4)
The addition of one of the fourdeoxynucleotide
triphosphates (dNTPs)(in the case ofdATP we add
dATPαS which is not a substrate for a luciferase)
initiates thesecond step.
DNA polymerase incorporates the correct,
complementary dNTPs onto the template.
This incorporation releases
pyrophosphate (PPi)
stoichiometrically.
ATP sulfurylase quantitatively converts PPi to
ATP in the presence of adenosine 5´
phosphosulfate.

11. This ATP acts as fuel to the
luciferase-mediated conversion of
luciferin to oxyluciferin that
generates visible light in amounts
that are proportional to the
amount of ATP.
 The light produced in the luciferase-catalyzed
reaction is detected by a camera and analyzed
in a program.
 Unincorporated nucleotides and ATP are
degraded by the apyrase, and the reaction
can restart with another nucleotide.
3 SOLiD SEQUENCING
SOLiD is an enzymatic method of sequencing
that uses DNA ligase, an enzyme used widely in
biotechnology for its ability to ligate double-
stranded DNA strands . Emulsion PCR is used to
immobilise/amplify a ssDNA primer-binding
region (known as an adapter) which has been
conjugated to the target sequence (i.e. the
sequence that is to be sequenced) on a bead.
These beads are then deposited onto a glass
surface − a high density of beads can be achieved
which which in turn, increases the throughput of
the technique.(Fig 5)

12. 
(Fig 5)