The advance version, sequences the whole genome efficiently with high speed and high throughput sequencing at reduce cost is termed as Next Generation Sequencing (NGS) or massively parallel sequencing (MPS).

1. THE FUTURE TOOL FOR CHOOSING DESIRSBLE
BREEDING SELECTIONS
NEXT GENERATION
SEQUENCING
Department of Genetics and Plant Breeding
Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur (M.P.)
CREDIT SEMINAR ON
Submitted by:- SHWETA TIWARI
En No.:- 190134008

2. INTRODUCTION
The procedure to determine the sequence of nucleotide in the fragment of DNA is termed as
DNA sequencing. While the advance version of high throughput sequencing method with more
accuracy, enhanced read length, increased speed, reduced time and diminishing cost is called
the Next Generation Sequencing.
The first generation sequencing in 1977 evolved into second generation sequencing in 2004, over a
span of ~30 years and in another 10 year we have third generation of sequencing developed.
FIRST GENERATION
SEQUENCING
SECOND GENERATION
SEQUENCING
THIRD GENERTAION
SEQUENCING
THE THREE ERA’S OF SEQENCING :-

3. DIFFERENCE BETWEEN
TRADITIONAL
SEQUENCING
METHODS
Although, they are distinct in
principle and procedure, but both of
them, separate in gel electrophoresis
on the basis of their lengths. The
sample is loaded into adjacent lane
of sequencing gel and the nucleotide
order is determined from
autoradiography image of the gel.
These are called First generation
sequencing.

4. 01. 700-1000bps at a time
07. Quality of sequence sacrificed :- Initial 15-20 sequence not precise and
degradation sequencing after 700bp.
06.Cost infectiveness:- $500 per 1000 bases, compared to less than 0.50 per 1000
bases for next generation sequencing.
03. Difficult to automate
02. Reduced resolution
05. More time requirement
04. Less accuracy
Need of NGS as Challenges Faced by
Traditional Sequencing Techniques

5. NEXT GENERATION
SEQUENCING
The Progressive and Improved Version
sequence the whole genome with high
speed and high throughput sequencing at
reduced cost is popularized as
NEXT GENERATION SEQUENCING
01. Also known as Massively Parallel
Sequencing
02. Faster, Cheaper and required much less
time in template preparation

6. NGS TIMELINE
454 Roche GS FLX
01
02
03
04
ILLUMINA Genome Analyzer
SOLiD Sequencer
The Ion Torrent & Pacific
Bioscience commercialized SMRT
2004
2006
2007
2010
05 2015Pocket-sized sensing device
MinION

7. Overview of General Procedure of Next Generation Sequencing
A.
D.
B.
C.
Emulsion PCR
Solid phase
amplification
NGS
Sequencing
and imaging
Data analysis
OVERVIEW OF NGS

8. 454 Sequencing
GS FLX, GS FLX Titanium series by 454 Life
Sciences/ Roche diagnostics Single Molecule Real Time (SMRT) Sequencing
By Pacific Bioscience,USA,
PACBIO RS
Helicos Genetic Analysis System
Helicos Tmgenetic Genetic Analysis System by
SeqLL,LLC
The SOLiD Sequencing
SOLiD 5500, SOLiD 5500XL, SOLiD 3 plus
Illumina Sequencing
Genome Analyzer, HiSeq, MiSeq,
NextSeq by Illumina, Inc.
Nanopore Sequencing Technologies
By Oxford Nanopore’s Complete Genomic by
Beijing Genomic Institude and GnuBIO by BioRad
SECOND GENERATION
SEQUENCING
THIRD GENERATION
SEQUENCING
Ion Torrent Sequencing
By Life Technologies
Ion Torrent PGM (Personal Genome Machine), Ion
Torrent Proton sequencing platform
CLASSIFICATION OF NEXT GENERATION
SEQUENCING

9. 454 DNA
SEQUENCING
0301
0402
In 2005 by 454 Life
sciences (now Roche
Diagnostics), USA.
First commercialized NGS Genome Sequencer
• GS FLX system
(~400bases)
• GS FLX Titanium series
(1Gb)
Currently available platform
One million beads at a
time and one run can
takes about 10h,
including template
preparation.
99.9% Accuracy
Processing
Sequencing by
synthesis :
PYROSEQUENCING
Principle
1. 454 DNA SEQUENCING

10. 1
Sensor record data based on
polymerization displayed in form
of pyrogram
Pyrogram Formed
2 1.LIQIUD PYROSEQUENCING
2. SOLID PYROSEQUENCING
Types
3 1. Inaccurate homo-polymer seq.
2. Shorter Read Length
3. Costly
Limitations
MECHANISM OF ACTION
Chemical luminescence enzymatic reaction with accuracy, flexibility, parallel processing and can
easily be automated. Incorporation of dNTPs, releases Pyrophosphate (PPi), which convert into
ATP, on whose presence luciferase convert luferin into oxyluciferin and illuminate the light.

11. 5tt
2. THE SOLID DNA SEQUENCING
THE SOLiD
SEQUENCING
0301
0402
The Applied Biosystem US,
commercialized the Polony
method in 2005 as SOLiD
3.0
COMMERCIALIZATION
Sequencing by
oligonucleotide
ligation detection
PRINCIPLE
The method is
developed by George
M. Church at Harvard
SCIENTIST
Trouble in sequencing
pallindromic
sequences
Short Read Length
LIMITATIONS
It is very cost effective with 0.13$ per
million bases. It read ~50 bases and
generate 20 Gb of total sequence per
run with time 6-7 days

12. PCR produced million
copies of template strand
which bound to large
polystyrene coated bead
and formed Polonies.
a & b
The template is
attached with bead
BEAD HYBRIDISATION
c & d
POLYMERASE COLONIES
e & f
Supernatant are
separated and attached
to slide
CENTRIFUGATION
AND ATTACHMENT
g
Base 1 & 2 are
complementary bases to
be sequenced while 3-5
are degenerated & base
6-8 are inosine base.
PROBE ANATOMY
h PRIMER BIND
Primer binding to
adapters through ligase

13. i& j
The probe anneal to primer
and fluorescence snapshot
PROBE ANNEALING & SNAPSHOT
k & l Dye ends cleaved and free 5`
phosphate for next round. The
entire process repeated four
times, each time with primer
offset by 1 bases
DYE CLEAVED OFF
m
WHOLE GENOME
SEQUENCING
n
PALLINDROMIC SEQUENCE
Palindrome sequences form
hairpin loop, which cause
difficulties in sequencing

14. ILLUMINA
SEQUENCING 0301
0402
Illumina,USA
commercialized the
Solexa NGS
technologyin 2007
Most widely used NGS
Illumina HiSeq
2000 and Illumina
GAIIx are market
leaders in 2011,
especially in
Europe and US
Currently available platform
Read length ranges
from 35 to150 bases
and accuracy is greater
than 98.5%
Processing
Sequencing by
synthesis
Principle
Also known as Bridge
amplification method.
In contrast to bead flow cell with
oligonucleotide adapters is used.
It reduced the cycle of
amplification
3. ILLUMINA SEQUENCING

15. ION
SEMICONDUCTOR
SEQUENCING
0301
0402
Commercialized as Ion
Torrent PGM (Personal
Genome Machine) and
Ion Torrent Proton
sequencing platforms.
COMMERCIALISATION
In illumine and other next
generation sequencing;
38- 40hr is required but
in this sequencing done in
only 3hr.
LESS TIME
Rapid Techniques as
sequencing is done in
real time with read
length of 200
nucleotides
PROCESSING
Sequencing by
synthesis
Principle
4. ION SEMICONDUCTOR SEQUENCING
Ion Torrent Sequencing,
pH mediated sequencing, Silicon
sequencing or Semiconductor
sequencing.
Also known as

16. MECHANISM OF ACTION
The method use semiconductor sensing device or ion chip senses the Hydrogen ions produce during
DNA synthesis by DNA polymerase. The change in pH is detected by sensing layer of micro well
translate the chemical signal into digital signal, measured within a second.

17. Features Roche (454) Illumina SOLiD
Chemistry Pyrosequencing (Sequencing
by synthesis)
Polymerase-based
(Sequencing by
synthesis)
Ligation-based
(Sequencing by
ligation)
Amplification Emulsion PCR Bridge Amp Emulsion PCR
Terminators Not Used Used Used
Detection based on Light emitted by luciferase Fluorescence from
flurophore
Fluorescence from
flurophore
Major error in base
calling
InDels Base substitution Base substitution
Chief cause of error Incorrect deduction of homo-
polymorphic length from
intensity of luminescence
Asynchronous DNA
synthesis in the later
cycle
Bias in fluorescence
intensities in later
machine cycle.
Template DNA
fragments attached
Beads in microtitier plate well A specific substrate on
flow cell
Beads in an
acrylamide matrix
Paired ends/sep Yes/3kb Yes/200bp Yes/3kb
COMPARISONS

18. Total sequence data/
run
400 Mb (GS FLX), ~1
Gb (GS FLX Titanium +)
400Gb (Hi seq 2000) 300 Gb (SOLiD
5500, SOLiD 5500
XL)
Time/run 7- 10 h 4- 8 days 5- 7days
Read length (short gun
sequencing)
400 bases (GS FLX+,
1000 bases)
35-150 bases (up to 250
bases by Hi Seq 2500)
35- 50 bases (SOLiD
3.0)
Read accuracy (%) 99.6 98.5 -
Template preparation Shotgun, paired end Shotgun, paired end paired end
Each base examined Once Once Twice
Improved base-calling
algorithm
Pyrobayes Ibis and BayesCall Rsolid
Draft genome
preparation
Yes Yes -
Cost per run (total) $8439 $8950 $17447
Cost per Mb $84.39 $5.97 $5.81
Current platforms GS FLX, GS FLX
Titanium
Genome Analyzer 1Gb, Hi
Seq 600 Gb
SOLiD 5500, SOLiD
5500 XL

19. The short read length that need to be assembled with the
help of various bioinformatics tools/pipelines into original
length template
LIMITATIONS OF SGS
01
PCR bias introduced by clone amplification, for detection of
base incorporation signal.
02

20. THIRD GENERATION SEQUENCING
The Third Generation of high throughput Sequencing develop as remedy to the
SGS limitations. Instead of sequencing clonally amplified template, single DNA
template is sequenced and this also need to minimal use of biochemical leading
to miniaturization of whole process to nano-scale.
PACIFIC BIOSCIENCE
SMRT HELICOS BIOSCIENCE,
USA
HELICOS
OXFORD NANOSPORE
TECHNOLOGY
NANOSPORE

21. Pacific Biosciences : Single Molecule Real Time (SMRT) Sequencing
1.Pacific Biosciences : Single Molecule Real Time
(SMRT) Sequencing
Phosphate linked nucleotide
Zero-mode waveguide
Two Technologies
1.
40kb but at 85% accuracy
Error rate 15%
Read length
2.
Allow multiplexing of1000
Of ZMW in parallel
High speed and High Fidelity
3.
PACBIO RS
Commercialized as
4.

22. 2.The Nanopore Sequencing
Oxford nanopore technology enables the identification of broad range of
analysis including DNA, RNA, Protein and monitor changes to an electrical
current as nucleic acid are passed through a protein nanopore.
ERROR RATE 4%
GridION
Optic signals
MiniION USB Stick

23. Conclusion
The advances in next generation sequencing revolutionized the
genetics and help in gaining more knowledge about the living
system and the phenotypes emerges out from the system. It has
provides great assistance in mastering the large-scale
information collection on living systems in diverse application
areasuch as treatment of human diseases, development of
alternative bio fuels, enhancement of crop yield, ensuring food
safety, forensics, etc. However, the First- and Second
generation sequencing facilitated, a complete understanding of
whole genome sequences and the information encoded therein,
a more complete characterization of the methylome and
transcriptome and a better understanding of interactions
between proteins. But the innovation of new generation single
molecule sequencing developed with potential for dramatically
longer readlengths, shorter time to result and lower overall cost
of DNA

24. NEXT GENERATION SEQUENCING
THE FUTURE TOOL FOR CHOOSING
DESIRSBLE BREEDING SELECTIONS

25. NEXT GENERATION SEQUENCING
THE FUTURE TOOL FOR CHOOSING
DESIRSBLE BREEDING SELECTIONS

27. THANKS