NEXT GENERATION

SEQUENCING
RIJA FATIMA (SP20-BSI-043)
MALEEHA HUSSAIN (FA20-BSI-073)
TABISH ALI (FA20-BSI-067)
 In 1953 James Watson and Francis Crick
described the double helix of DNA. In 1977
Frederick Sanger invented a method for
sequencing the DNA sequence using

INTRODUCTIO dideoxynucleosides.

N:
Through this method and via using
fluorescence tagged nucleotides and
chromatography, sequencing 600-800 bp
sequences is possible.
 In 1960 Thomas Dale Brock
reported
hyperthernmophiles living PCR (Polymerase Chain
in hot springs at Reaction) was Develop in
Yellowstone National Park. 1983 by Kary Mullis.

1960 1983

CONT..
1976

After that in 1976 Chien et

al isolated thermostable
DNA polymerase Thermus
aquaticus, and now we use
it for PCR
 Next-generation sequencing (NGS) is a massively parallel
sequencing technology that offers ultra-high throughput,
scalability, and speed. The technology is used to determine
the order of nucleotides in entire genomes or targeted
regions of DNA or RNA.

WHAT IS NEXT NGS has revolutionized the biological sciences, allowing

GENERATION labs to perform a wide variety of applications and study
biological systems at a level never before possible.
SEQUENCING:
WHY THERE IS A NEED OF NEXT
GENERATION SEQUENCING:

• Before the advent of NGS, the Sanger sequencing technique

was used to determine the DNA sequence. However, due to
the limitations of Sanger sequencing in throughput and
relatively high costs, it was difficult to sequence a large
DNA/RNA sample. The NGS technology was developed to
overcome the shortcomings of Sanger sequencing. NGS
technologies can parallelly sequence thousands of DNA
molecules simultaneously with high throughput and speed
NGS TOOLS:

• Visualization
• Integrative Genomics Viewer (IGV)
• Artemis
• Abrowse
• Integrated Genome Browser (IGB)
 NGS
MACHINES:
 The growing power and reducing cost
sparked an enormous range of application
of next generation sequencing( NGS)
technology
NGS platform was developed and put on the
DEVELOPMEN market by Roche in 2005
T OF NGS:
The development of the next generation
sequencing technology has contributed to
the trend sustaintially by reducing cost and
producing massively sequencing data
STEPS IN NEXT-
GENERATION
SEQUENCING (NGS):
• Next-generation sequencing is mainly
performed in three steps.
• 1. Library preparation: using the
random fragmentation of DNA and the
following ligation with custom linkers
library is prepared.

• 2. Amplification: PCR and clonal

amplification methods are used for the
amplification of the library.
• 3. Sequencing: there are different
sequencing methods, and sequencing is
done using any one of the methods.
APPLICATIONS OF NGS:

Whole Genome Sequencing (to find point mutation or be sure about gene integration in right place)
•Target Sequencing (hotspot sequences mutation for cancer or immune system disease)
•De Novo Sequencing and Assembly (for new organism which have not enough information about them)
•RNA-Sequencing (to detect coding and non-coding sequences and sometime we can use it as genome
sequence)
•Epigenetic changes
 • Single cell sequencing
• •Free DNA sequencing (detection of cancer or genetic disorders
before birth)
CONT… • *Long non-coding RNA interactions (fore gene translation
regulation)
• •For Methylation Assisted Isolation of Regulatory Elements
(AIREDNase sequencing)
WHAT CAN NEXT
GENERATION SEQUENCING
DO:

• Detects nucleotide substitution

• Detects indel
• Detects CNV
• Detects translocation
• Detects inversion
• Detects methylation
CONT…..

• Provides cheaper and high throughput alternatives to DNA

sequencing.
• Facilitates the discovery of genes and regulatory elements
associated with diseases.
 ILLUMINA SEQUENCING:
• ln NGS, vast numbers of short reads are sequenced in a single stroke.
• To do this, firstly the input sample must be cleaved into short sections. The length of these sections
will depend on the particular sequencing machinery used.
• In Illumina sequencing, 100-15Obp reads are used. Somewhat longer fragments are ligated to
generic adaptors and annealed to a slide using the adaptors. PCR is carried out to amplify each read,
creating a spot with many copies of the same read. They are then separated into single strands to be
sequenced.
 454 SEQUENCING:
• Roche 454 sequencing can sequence much longer reads than llumina. Like ilumina, it does this by
sequencing multiple reads at once by reading optical signals as bases are added.
• As in Illumina, the DNA or RNA is fragmented into shorter reads, in this case up to 1kb. Generic
adaptors are added to the ends and these are annealed to beads, one DNA fragment per bead. The
fragments are then amplified by PCR using adaptor-specifc primers.
• Each bead is then placed in a single well of a slide. So each well will contain a single bead, covered in
many PCR copies of a single sequence. The wells also contain DNA polymerase and sequencing
buffers.
 ION TORRENT: PROTON/PGM REQUENCING
• Unlike Illumina and 454, lon torrent and lon proton sequencing do not make use of optical signals.
Instead, they exploit the fact that addition of a dNTP to a DNA polymer releases an H+ ion.
• As in other kinds of NGS, the input DNA or RNA is fragmented, this time ~200bp. Adaptors are
added and one molecule is placed onto a bead. The molecules are amplified on the bead by
emulsion PCR. Each bead is placed into a single well of a slide
TYPES OF NEXT GENERATION SEQUENCING:

1: Target sequencing:
• Allows identification of disease causing mutations.
• Diagnosis of pathological conditions.
2: RNA Sequencing:
• Provides information on the entire transcriptome of a sample in a single analysis.
• Acts as a strong alternative to the use of microarrays in gene expression studies.
LIMITATIONS OF NEXT GENERATION
SEQUENCING:

• Expensive
• Inaccurate sequencing of homopolymer regions
• Sequence errors.
D/F BETWEEN DNA SEQUENCING AND NGS :

DNA SEQUENCING = DETERMINATION OF PRECISE ORDER NEXT GENERATION SEQUENCING= HIGH THROUGHPUT
OF NUCLEOTIDES WITHIN A DNA MOLECULE SCREENING METHOD MASSIVE PARALLEL SEQUENCING
DURING WHICH MILLIONS OF FRAGMENTS OF DNA FROM
A SINGLE SAMPLE ARE SEQUENCED IN UNISON
WORK FLOW OF
NGS:

The next-generation sequencing

workflow contains three basic steps: 
• Lab preparation.
• Sequence.
• Analyze.
LAB
PREPARATION:
• Library preparation is crucial to the
success of your NGS workflow. This
step prepares DNA or RNA samples to
be compatible with a sequencer.
Sequencing libraries are typically
created by fragmenting DNA and adding
specialized adapters to both ends. In the
Illumina sequencing workflow, these
adapters contain complementary
sequences that allow the DNA
fragments to bind to the flow cell.
Fragments can then be amplified and
purified.
 SEQUENCE:
• During the sequencing step of the NGS workflow, libraries are loaded onto a flow cell and placed on
the sequencer. The clusters of DNA fragments are amplified in a process called cluster generation,
resulting in millions of copies of single-stranded DNA. On most Illumina sequencing instruments,
clustering occurs automatically.
DATA ANALYZE:

• After sequencing, the instrument software identifies nucleotides (a process called base calling) and the
predicted accuracy of those base calls. During data analysis, you can import your sequencing data into a
standard analysis tool or set up your own pipeline.
 Features of NGS data

Short sequence reads

NGS ~500 bp: 454 (Roche)

TECHNOLOGI
35 – 150 bpSolexa(Illumina), SOLiD(AB)
ES:
Huge amount of sequence per run

Gigabases per run (600 Gbp for

Illumina/HiSeq2000)
 Huge number of reads per run

Up to billions

Bias against high and low GC content (most

platforms)
CONT…
GC% = (G + C) / (G + C + A + T)

Higher error (compared with Sanger)

Different error profiles

 The main disadvantage of NGS in the clinical setting is
putting in place the required infrastructure, such as
computer capacity and storage, and also the
personnel expertise required to comprehensively
analyse and interpret the subsequent data.All
second-generation NGS technologies are dependent
on amplification before sequence analysis.

This amplification step is needed to generate a large

DISADVANTA enough number of copies of each DNA template so
that there is sufficient signal strength for each base
addition.Three different techniques are commonly
GS OF NGS: used to amplify DNA fragments before sequencing:
emulsion PCR, bridge amplification and DNA nanoball
generation.

Second-generation NGS technologies can be

categorized based on what DNA amplification
techniques they utilize.