Next generation sequencing (NGS) refers to modern DNA sequencing technologies that allow for high-speed, low-cost sequencing of entire genomes. NGS works by massively parallel sequencing of millions of DNA fragments. The Illumina sequencing by synthesis method is the most commonly used NGS approach. It involves library preparation, cluster generation on a flow cell, sequencing via reversible dye-terminator chemistry, and computational analysis of sequenced reads. Key advantages of NGS include its scalability, unlimited dynamic range, tunable coverage levels, and ability to multiplex many samples simultaneously in a single run.

1. NEXT GENERATION SEQUENCING (NGS)

2. INTRODUCTION
TO
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SEQUENCING------S

3. DNA SEQUENCING
 DNA sequencing is the process of
determining the precise order
of nucleotides within a DNA molecule.
 It includes any method or technology
that is used to determine the order of the
four bases—
adenine, guanine, cytosine,
and thymine—in a strand of DNA.

4. DNA SEQUENCING METHODS
 CLASSICAL METHODS:
1. Sanger Sequencing Method
2. Maxam And Gilbert Sequencing Method
Maxam–Gilbert sequencing along with the Sanger
method, represents the first generation of DNA
sequencing methods.

5.  Developed by Fredrick Sanger and his colleagues in 1977
 It is a method of DNA sequencing based on the selective
incorporation of chain-terminating dideoxynucleotides by
DNA polymerase,
 With the advent of the Sanger method scientists gained the
ability to sequence DNA in a reliable, reproducible manner.
 However, his method limitations showed a need for new and
improved technologies for sequencing large numbers of human
and other genomes.
SANGER SEQUENCING

6. SANGER SEQUENCING

7. MAXAM AND GILBERT SEQUENCING METHOD
 Developed by Allan Maxam and Walter Gilbert in 1976–
1977.
 This method is based on nucleobase-specific partial
chemical modification of DNA and subsequent cleavage
of the DNA backbone at sites adjacent to the
modified nucleotides.
 Maxam–Gilbert sequencing is no longer in widespread
use, having been supplanted by next-generation
sequencing methods.

8. MAXAM AND GILBERT SEQUENCING METHOD

9.  In the late 20th and early 21st century, efforts
have been made towards the development of new
methods to replace“first-generation” technology.
 The newer methods are referred to as next-
generation sequencing (NGS) and their use has
changed the scientific approaches in both basic
and applied research in many of scientific
disciplines.

10. NEXT-GENERATION DNA SEQUENCING
 Next generation sequencing (NGS) is a new method for
sequencing genomes at high speed and at low cost.
 It is also known as second generation sequencing (SGS)
or massively parallel sequencing (MPS).
 It is the catch-all term used to describe a number of different
modern sequencing technologies including:
 Illumina (Solexa) sequencing ( Shankar
Balasubramanian and David Klenerman in 1998)
 Pyrosequencing (454)
 Ion torrent sequencing (2011)
 SOLiD sequencing

12. NEXT
GENERATION
SEQUENCING

13. THE BASICS OFNEXT
GENERATION
SEQUENCING

14. PRINCIPLE
 the concept behind NGS technology is similar to
CE sequencing.
 DNA polymerase catalyzes the incorporation of
fluorescently labeled deoxyribonucleotide
triphosphates (dNTPs) into a DNA template
strand during sequential cycles of DNA
synthesis.
 During each cycle, at the point of incorporation,
the nucleotides are identified by flourophore
excitation.

15. PRINCIPLE
 The critical difference is that, instead of
sequencing a single DNA fragment, NGS
extends this process across millions of fragments
in a massively parallel fashion.
 More than 90% of the world's sequencing data
are generated by Illumina sequencing by
synthesis (SBS) chemistry.
 It delivers high accuracy, a high yield of error-free
reads.

16. 4 BASIC STEPS
 1. Library Preparation—
 Fragmentation
 Adopter ligation reactions
 PCR amplification
 Gel purification.

18. 2. CLUSTER GENERATION
 For cluster generation, the library is loaded
into a flow cell where fragments are captured
on a lawn of surface-bound oligos
complementary to the library adapters. Each
fragment is then amplified into distinct, clonal
clusters through bridge amplification . When
cluster generation is complete, the templates
are ready for sequencing.

20. 3. SEQUENCING
 Illumina SBS technology uses a proprietary
reversible terminator– based method that
detects single bases as they are incorporated
into DNA template strands . As all four
reversible terminator–bound dNTPs are
present during each sequencing cycle, natural
competition minimizes incorporation bias and
greatly reduces raw error rates compared to
other technologies. The result is highly
accurate base-by-base sequencing that
virtually eliminates sequence context–specific
errors, even within repetitive sequence
regions and homopolymers.

22. 4. DATA ANALYSIS—
 During data analysis and alignment, the
newly identified sequence reads are aligned
to a reference genome . Following alignment,
many variations of analysis are possible,
such as single nucleotide polymorphism
(SNP) or insertion-deletion identification,
read counting for RNA methods,
phylogenetic or metagenomic analysis, and
more.

26. ADVANCES IN SEQUENCING TECHNOLOGY

27. PAIRED-END (PE) SEQUENCING
 PE sequencing involves :
1. The double strand DNA get linear into forward
and reverse.
2. The original forward strand is cleaved and
wash off. Read 2 primer get attach to reverse
strand.
3. Sequencing steps are repeated as addition of
dNTPs and blinking of light. Read 2 product is
wash away. The entire process produce
millions of reads representing all the fragments

28. PAIRED-END (PE) SEQUENCING
4. Sequences from pool sample library are
separated based on the unique intensities
introduce during the sample preparation.
5. From each sample, reads with similar
stretches are clustered and forward and
reverse reads are prepared having
contagious sequencing.
6. These contagious sequences are then align
back to reference genome.

30. PAIRED-END (PE) SEQUENCING
 Advance in NGS technology occurred with the
development of paired-end (PE) sequencing
 PE sequencing involves :
1. Sequencing both ends of the DNA fragments in a
library.
2. Aligning the forward and reverse reads as read pairs.
3. To produce twice the number of reads for the same
time.
4. Effort in library preparation in which , sequences
aligned as read pairs enable more accurate read
alignment and the ability to detect index.

33. UNLIMITED DYNAMIC RANGE
 The digital nature of NGS allows a unlimited
dynamic range for read-counting methods,
such as gene expression analysis.
 NGS quantifies discrete, digital sequencing
read counts.
 Researchers can tune the sensitivity of an
experiment by increasing or decreasing the
number of sequencing reads.

34. UNLIMITED DYNAMIC RANGE
 Researchers can quantify subtle gene
expression changes with much greater
sensitivity.
 Sequencing runs can be zoom in with high
resolution on particular regions of the
genome, or provide a more expansive view
with lower resolution.
 Because the dynamic range with NGS is
adjustable and nearly unlimited.

35. TUNABLE COVERAGE
 NGS easily tune the level of coverage.
 For example:
 Somatic mutations only exist within a small
proportion of cells in a given tissue sample.
Using mixed tumor–normal cell samples, the
region of DNA having mutation must be
sequenced at extremely high coverage, often
upwardsof1000×, to detect these low frequency
mutations within the mixed cell population

36. TUNABLE COVERAGE
 Targeted sequencing allows you to focus
your research on particular regions of the
genome.
 We can do a shallow scan across multiple
samples .
 Also can sequence at greater depth with
fewer samples to find rare variants in a
given region.

39. ADVANCE LIBRARY PREPARATION
 The first NGS library prep protocols
involved:
1. DNA fragmentation and target selection:
Take DNA and cut into smaller pieces by
physical or enzymatically. This called
fragment library. Or amplify the desired
fragment through PCR of known
sequence. This will call amplicon library.

40. ADVANCE LIBRARY PREPARATION
2. Adapter sequences: Adapters are added
to the both ends of fragment. Of 20-40bp
of known sequences.
3. Size selection: Separate the adapter
linked fragments on gel according to
sizes. The band corresponding the size of
interest is collected.

41. ADVANCE LIBRARY PREPARATION
4. Final library quantification and QC:
Commonly methods used are:
 Bioanalyser system : this method give
you both library concentration and
fragment size information.
 qPCR: this method provide most
accurate lib quantification. But lack
fragment size info.

42. ADVANCE LIBRARY PREPARATION
 Nextera® XTDNA Library Preparation,
current NGS protocols have reduced the
library prep time to less than 90minutes.
 PCR-free and gel-free kits are also available
for sequencing methods.
 PCR-free library preparation kits result in
superior coverage of traditionally challenging
areas such as high AT/GC-rich regions,
promoters, and homopolymeric regions

43. MULTIPLEXING
 NGS has multiplexing ability. Multiplexing
allows large numbers of libraries to be
sequenced simultaneously during a single
sequencing run. Reducing your cost for
sample.
 With multiplexed libraries, unique index
sequences are added to each DNA
fragment during library preparation so that
each read can be identified and sorted
before final data analysis.

44. MULTIPLEXING
 Library preparation:
Index sequences are added to the DNA or
RNA library to be sequence. Each sample
has unique sequence that allow the software
to identify and group the sequencing reads
from each sample.

46. MULTIPLEXING
 How does one calculate how many samples to
multiplex on sequencing run?
 There 3 main things we need to know before
getting started:
1. Sequencer throughput(Gb) throughput ability of
sequencer platform.
2. Required sequencing coverage: for desired
sample.
3. Size of Genomic targets (Gb): the size of genome
to be sequence.
Throughput method, in which a single 10-hour run
of a machine can produce 400 million or more
base pairs of DNA sequence information.

47. MULTIPLEXING
 Then use simple equation:
 #samples to multiplex – Sequencer
throughput/coverage *size of genomic
targets
 This estimate the number of samples we
can multiplex.

48. MULTIPLEXING
 With PE sequencing and multiplexing, NGS
has dramatically reduced the time to data for
multi sample studies and enabled
researchers to go from experiment to data
quickly and easily.
 Demultiplexing is a process in which
sequencing reads from pooled libraries are
identified and sorted computationally before
final data analysis

49. SCALABLE INSTRUMENTATION
 NGS technology is also highly flexible and
scalable.
 Sequencing systems are available for every
method and scale of study, from small
laboratories to large genome centers.
 Illumina NGS instruments range from the bench
top MiniSeq™ System, to the
NovaSeq™6000System, having scalable
throughput ability and flexibility for virtually any
sequencing method, genome, and scale of
project.

51. NOVASEQ™6000SYSTEM

52. FLEXIBLE, SCALABLE INSTRUMENTATION
 The new Nova Seq Series of systems unites the
latest high-performance imaging with the next
generation of Illumina patterned flow cell
technology to deliver massive increases in
throughput.
 This flexibility allows researchers to configure
runs tailored to their specific study requirements,
with the instrument of their choice.
 Flexible run configurations are also engineered
into the design of Illumina NGS sequencers.