Next-Generation Definition:

Sequencing 1. Next-generation sequencing (NGS) is a type of DNA sequencing

technology that uses parallel sequencing of multiple small fragments
of DNA to determine sequence

2. Next-generation sequencing (NGS) ----the catch-all term used to

describe a number of different modern sequencing technologies
including:

a. Illumina (Solexa) sequencing

b. Roche 454 sequencing
c. Ion torrent: Proton / PGM sequencing
d. SOLiD sequencing
i. 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
revolutionised the study of genomics and molecular
biology.
3. Amplification based ---- have been referred to as "second-
generation"
4. single-molecule sequencing technologies referred to as "third-
generation"

Next-Generation 5. Next-generation sequencing (NGS), also known as high-

Sequencing throughput sequencing
6. The principle behind Next Generation Sequencing (NGS) is similar to
that of Sanger sequencing, which relies on capillary
electrophoresis.
7. "Next-generation" and "massive-parallel" DNA sequencing are
blanket terms used to refer collectively to the high throughput DNA
sequencing technologies available which are capable of sequencing
large numbers of different DNA sequences in a single reaction (i.e.,
in parallel).
 8. Beginning in 2005
9. In massively parallel sequencing, the process is a stepwise reaction
series that consists of
a. nucleotide addition step,
b. detection step that determines the identity of the incorporated
nucleotides on each fragment focus being sequenced, and
c. wash step that may include chemistry to remove fluorescent
labels or blocking groups.
i. next-generation sequencing instruments conduct
sequencing and detection simultaneously rather than as
distinct processes, one of which is completed before
the other takes place.

The four main advantages of NGS over classical Sanger

sequencing are:
1. Speed
2. cost
3. sample size
4. accuracy

NGS is significantly cheaper, quicker, needs significantly less

DNA and is more accurate and reliable than Sanger sequencing
NGS is massively parallel, allowing 300 Gb of DNA to be read on
a single run on a single chip.
Limitations
1. The increased throughput of NGS reactions comes at the cost of read
length, as the most readily available sequencing platforms (Illumina,
Roche, SoLiD) offer shorter average read lengths (30400 bp) than
conventional Sanger-based methods (5001 kb;
Steps Involved in Next generation methods of DNA sequencing
1. Source: DNA or RNA Extraction
2. Genome Fragmentation
3. End Repair& Adapter Ligation
 4. Surface Attachment
5. Amplification Creation of Library [could include Bar coding]
6. Sequencing----
a. Single end/Paired end
b. Sequencing Massively Parallel Sequencing
Sequencing by synthesis
Sequencing by ligation
7. Reassemble & Analysis
a. Bioinformatics - Data Analysis .
Library Preparation DNA samples are randomly fragmented and
1. Genome Fragmentation platform-specific adaptors are added to the
2. End Repair& Adapter Ligation flanking ends to produce a library.
3. Amplification Library is then amplified through PCR.
4. (Platformspecific amplification e.g. beads or
glass)
Genome Fragmentation Firstly, DNA is fragmented either enzymatically
or by sonication (excitation using ultrasound) to
create smaller strands called as Reads
End Repair& Adapter Ligation

Emulsion PCR (emPCR)

Amplification 1. DNA fragmentations and adaptor ligation.
Current NGS platforms utilize clonal 2. DNA fragments are added to an oil mixture
amplification on solid supports via two containing millions of beads.
main methods: 3. Emulsion PCR results in multiple copies of the
emulsion PCR (emPCR) fragment.
bridge amplification (DNA cluster 4. Beads are deposited on plate wells ready for
generation) sequencing.

bridge amplification (DNA cluster generation)

In Illumina Sequencing
1. DNA fragmentations and adaptor ligation
2. The DNA is then attached to the surface of the
cell at random where it is exposed to reagents for
 polymerase based extension.
3. On addition of nucleotides and enzymes, the
free ends of the single strands of DNA attach
themselves to the surface of the cell via
complementary primers, creating bridged
structures.
4. Enzymes then interact with the bridges to make
them double stranded, so that when the
denaturation occurs, two single stranded DNA
fragments are attached to the surface in close
proximity.
5. Repetition of this process leads to clonal
clusters of localised identical strands.
6. In order to optimise cluster density,
concentrations of reagents must be monitored
very closely to avoid overcrowding.
Sequencing Platforms Popular Platforms:
Roche 454 ,
AB SOLiD, Illumina(HiSeq,MiSeq).
Newer Platforms (Third Generation):
1. Ion Torrent, PacBio RS, Oxford
Nanopore.
Pyrosequencing Pyrosequencing is based on the 'sequencing by
synthesis' principle, where a complementary
strand is synthesised in the presence of
polymerase enzyme .
Pyrosequencing detects the release of
pyrophosphate when nucleotides are added to
the DNA chain.

Pyrosequencing:

Applications
isothermal amplification instead of emulsion
Illumina/Solexa
1. Most popular sequencing
technique nowadays!
2. , 100-150bp reads are used.
3. longer fragments are ligated to
generic adaptors and annealed to a
slide using the adaptors.
4. PCR Amplification by Bridge
Amplification
5. This technique was pioneered by
Illumina, with their HiSeq and
MiSeq platforms.
a. HiSeq is the cheapest of
the second generation
sequencers with a cost of
$0.02 per million bases
b. . It also has a high data
output of 600 Gb per run
1. non-electrophoretic,
2. bioluminescence method that measures the
release of inorganic pyrophosphate by
proportionally converting it into visible light
using a series of enzymatic reaction
3. Nucleotide incorporation generates light seen as
a peak in the Pyrogram trace
1. Whole genome sequencing
2. Targeted resequencing
3. Sequencing-based
4. Transcriptome Analysis
5. Metagenomics
1. Solid-phase amplification
2. can produce 100-200 million spatially separated
clusters, providing free ends to which a
universal sequencing primer can be hybridized
to initiate the NGS reaction
3. Reversible terminator sequencing method is used by
Illumina/ in which modified nucleotides are
used in reversible termination.
4. Reversible termination uses bridge PCR,
improving the efficiency of this stage of the
process.
a. Reversible terminators can be grouped
into two categories:
i. 3-O-blocked reversible
terminators and
ii. 3-unblocked reversible
terminators.
 which takes around 8 days
to complete.

Sequencing by Cyclic Reversible Termination
(CRT):
CRT uses reversible terminators in a cyclic
method that comprises
1. nucleotide incorporation,
2. fluorescence imaging and
3. cleavage

nucleotides that have not been 4. DNA polymerase, bound to the primed
incorporated are washed away and the
template, adds or incorporates just one
fluorescent branch is cleaved using TCEP
fluorescently modified nucleotide
(tris(2-carboxyethyl)phosphine). TCEP also
removes the 3-O-azidomethyl group,
5. Unincorporated nucleotides are washed
regenerating 3-OH, and the cycle can be
away and a four-color imaging is
repeated
acquired by total internal reflection
fluorescence (TIFR) using two laser 3

6. A cleavage step (TCEP, a reducing

agent)---- (tris(2
carboxyethyl)phosphine) removes the

a. the fluorescent dye

b. the 3-O-azidomethyl group,
regenerating 3-OH, and the cycle
can be repeated