NGS

Outline
• Timeline of NGS platform
• Introduction
• First generation sequencing
• NGS work flow
• NGS Platforms
• Application of NGS
• Future challenges
• Conclusion
Timeline of DNA sequencing technologies and platforms
 Introduction
“Knowledge of sequences could contribute much to our understanding of living matter”-
Sanger

DNA sequencing -process of determining the precise order of nucleotides

Next-generation sequencing
– High-throughput approach
– Sequence several millions to billions DNA fragments in massively parallel
manner nature.com/subjects/ngs

Why NGS ????

– Sample preparation - faster and straightforward compared to sanger sequencing
– Capability to generate massive volume of data
– Accurate, fast and inexpensive method
– Overcomes the limitation of microarrays to detect poorly expressed genes
– More than a billion short reads are obtained in single run
(Reis-filho, 2009)
Next generation sequencing platforms
 Sanger sequencing
• ddNTPs – Chain Termination
• Sanger sequencing (1977)
– Chain termination method
– Sequencing by synthesis
– Dideoxy sequencing
– Most common approach used
for DNA sequencing
– Nobel prize – 1980
– Considered as the GOLD
STANDARD
 Maxam - Gilbert sequencing ((1977)
Chemically cleavage Method
 No need of DNA synthesis
 Treatment of DNA with certain
chemicals  DNA cuts into
fragments  monitoring of
sequences
 NGS Workflow
• Source of sample (DNA, RNA)
Sample • Qualify and quantify samples

• Prepare platform specific library

Library • Qualify and quantify library

Sequenc • Perform sequencing run reaction on NGS platform

ing

Analysis
• Application specific data analysis pipeline

Grada et al.;2013
 Cluster Amplification: Bridge PCR
Used by Illumina

– DNA fragments are flanked with adaptors

– Flow cell is coated with primers complementary to the two adaptor sequences

– Isothermal amplification

– Clusters of DNA molecules are generated on the chip

– Each cluster is originated from a single DNA fragment, and is thus a clonal
population
 Cluster Amplification :Emulsion PCR
Used by SOLiD, Ion torrent and pyrosequencig
– Fragments attached with adaptors
– One PCR primer is attached to the surface of a bead
– DNA molecules are amplified on the beads within a water –oil emulsion
– Each bead bears clonal DNA originated from a single DNA fragment
– Beads placed into the wells of sequencing chips
– One well, one bead
 Roche (454) GS FLX sequencer
Principle

–Based on pyrosequencing
principle

–Sequencing by synthesis

–Detection of released
pyrophosphate

Procedure
1. DNA fragmentations and adaptor
ligation
2. Emulsion PCR
3. Beads are placed into wells of
PTP
4. Slide is flooded with any one of
the four nucleotide Light signal recorded by camera
(Metzker et al., 2010)
 Illumina Solexa Analyzer

• Major NGS platforms

• Reads of 100-150bp
• Chemistry
– Fragment of DNA
– Sonication
– Nebulization
– RE digestion
• Library preparation
– Bridge amplification
• Sequence by synthesis
• Data analysis

http://res.illumina.com/documents/products/techsp
 Illumina Solexa Analyzer

Determine first base

• Through CRT strategy

• Which sequences the template strand one nucleotide at a time through

progressive rounds of base incorporation, washing, imaging

• Fluorescently labeled 3’-o-azidomethyl-dNTPs are used to pause the reaction,

enabling removal of unincorporated bases and fluorescent imaging to determine
the added nucleotide

• Scan the flow cell with a CCD camera,

the fluorescent moiety and the 3’ block

are removed, and the process is repeated

(Guo et al., 2008)

http://res.illumina.com/documents/products/techsp
Illumina Solexa Analyzer

http://res.illumina.com/documents/products/techsp
 Applied Biosystems SOLiD Sequencer
• Sequencing by oligonucleotide ligation
and detection

• Ligase enzyme used instead of

polymerase

• Procedure

1. Sample preparation

2. Emulsion amplification

3. Ligation reaction and imaging

• Sequencer adopts the technology
of two-base sequencing based on
ligation sequencing
• Each sequencing involves 5 rounds
of cyclic steps
4. Data analysis Mardis;2013
Ligation and Imaging…Ligation chemistry

Mardis;2013
Ligation and Imaging…Ligation chemistry

Mardis;2013
Data analysis

Mardis;2013
 Ion Torrent PGM sequencer
• Sequencing platform does not uses optic signals

• Clustal amplification- emulsion PCR

• Concept

– Addition of a dNTPs to a DNA, polymer releases an

H+

– Change in pH is detected by the semiconductor

installed in each well of the chip

– Each time the chip was flooded with one nucleotide

after another, if it is not the correct nucleotide, no
voltage will be found

– Voltage change corresponding to each type of

nucleotide in different way

• Run time: 3 h Mardis;2013

 Helicos True Single molecule sequencing
• Considered as TGS

• First NGS platform

which used the concept of
single molecule
fluorescent sequencing

• No amplification needed

• Process
•DNA fragmentation
•Tailed with poly A
• Hybridized to a flow
cell surface containing
oligo-dT
•Sequencing-by-
synthesis of billions of
molecules in parallel (Thompson et. al., 2010)
 Pacific Biosciences(SMRT™)
The SMRT - considered as TGS

• Zero-mode waveguides (ZMW) ZMW

– Heart of the technology

– Detect world’s smallest light
volume
DNA polymerase
• Each SMRT contain tens of thousand
ZMW

• DNA template- polymerase complex

are immobilized at bottom of ZMW

• Four dNTPs labelled with different

dyes are added

• Incorporation of nucleotides
produces signal
Metzker,M.L.(2010)
 Nanopore DNA sequencing
• 4th -generation
• Smallest Sequencer
• Base detection without labels
• Ist hand held nanopore DNA sequencer
• Principle
– DNA strand is pulled through the
nanopore by the enzyme

– Passing of molecule through the pore

causes a temporary change in the
potential between the two
compartments for identification of
specific molecule
• Types
Biological nanopore- transmembrane
protein channels
Example: alpha-hemolysin,
Solid-state nanopore
Example MinION (Wang et al., 2015)
 Comparisons sequencing platforms
Platform Amplificati Sequencing Signal Run time Advantage Disadvantage Accuracy
on method chemistry detection
method
Sanger Vector Chain Long read Expensive 99.9%
sequencing based termination Impractical for
larger genome
seq
454gsflx Emulsion Pyrosequenc Light 23h Long read Expensive 99.9%
(Roche) PCR ing Homopolymer
error
Illumina( hi Bridge PCR Sequencing Light 2 days Hts Short read 98%
seq, mi seq) by synthesis Expensive Long run time
AB solid Emulsion Sequencing Light 8 days Low cost / base Short read 99.9%
PCR by ligation Long run time

Ion torrent Emulsion Sequencing Ph 3h Short run time Homopolymer 99.9%

PCR by synthesis Less expensive error

Longer read
lengths

Helicos No PCR TSMS 1gb/h No PCR 100%

Pacbio No PCR SMRT Light 20 min Longest read Low yield at 100%
SMRT Fast high accuracy
No PCR Expensive
http://www.molgen.mpg.de/899148/ows2013
 Applications of NGS

NEXT GENERATION SEQUENCING

TRANSCRI EPIGENOM METAGENO
GENOMICS
PTOMICS ICS MICS

Whole genome
seq RNA- seq ChIP-Seq
Exome seq Targeted RNA Methyl-Seq Microbiome
De novo seq seq Ribosome Seq
Targeted seq Nc RNA seq profiling
Three NGS levels: Which sequencing level should we
choose?
GENE EXPRESSION PROFILING THROUGH
RNA-seq
CELLS ISOLATED(PBM’C)

INFECTED/CHALL
CONTROL
ENGED

RNA SEQ OF RNA Seq OF

CONTROL INFECTED

READS READS
GMAP

MAPPED READS

RNA-SEQ DATA ANALYSIS USING CUFFLINKS

PACKAGE
(cufflink, cuffmerge, cuffdiff)

DETECTION OF DIFFERENTIALLY EXPRESSED

GENES

IDENTIFICATION OF GENES
UP OR DOWN REGULATED
SAGE (Serial Analysis of Gene Expression)

Velculescu V.E.(1995),Science 270:484-487

 Noncoding RNA Profiling and Discovery

• ncRNA – RNA that are not translated into protein product.

• Includes tRNA, rRNA, snRNA, snoRNA, siRNA, miRNA

• siRNA & miRNA- Posttranscriptional regulation of gene expression

• MPSS highly efficacious for discovery of novel miRNA

• Sequence based approaches helps in detection of variants of known

miRNA
 Epigenetic Modification Analysis

• Epigenetics – study of heritable gene expression that does not involve DNA
sequence.

• Two major type of modifications

– DNA methylation

– Histone tail modification

• Power of NGS gave a boost to the study of (genome wide) DNA methylation

• NGS -analysis clearly reveals all sequences , besides enriching methylated DNA
sequences

• Chemical tricks can be used to identify methylated nucleotides.

• Sequencing both untreated and bisulphate-treated DNA will highlight the c-

nucleotides that are methylated and not chemically converted resulting in a t when
sequenced
Ancient DNA
• Usually highly fragmented with average fragment lengths 51.3 bp
• Isolated from fossils
• Contains very different levels of contamination
• Difficult to sequence
 NGS technologies an ideal tool for ancient DNA research
 Large data generated by NGS
 Short read length (Knapp and Hofreiter, 2010)
• Uncovering the potential
cause of the disappearance
of the honeybee

• To detect the cause of an

infectious disease
Cox-Foster,2007
 Whole Genome Sequencing

• Whole genome de novo sequencing

– Process sequencing fragments of DNA and
assembling them to make a full-length genome
without referring to any previous information of
available sequences for the same species
• Comparison between cynomolgus(CE) and Chinese
rhesus(CR) macaques
• Abundant genetic heterogeneity
• Whole genome sequencing helps to quantify
introgression influence
• High degree of sequence similarity with human disease
gene orthologs
Guangmei Yan et. al (2011), Nature Biotechnology; p:1019-1023
 Genomic prediction in livestock using NGS
• NGS- explore relationship between genetic and phenotypic diversity with
high resolution

https://doi.org/10.12972/jabng.2017
 Selection of Disease Resistant Animals For Breeding

• Disease resistance and immune response are quantitative traits

• Direct selection based on recovery after infection is not a practical way of disease
resistance breeding

• Focusing on one bacteria or virus cannot improve the disease resistance to multiple
pathogens

• Genetic basis of general resistance to multiple infectious diseases, and

identification of indicator traits that can be used in breeding programme

• Two groups : 1. Not infected(control)

2.Infected or challenged/immunised/cell culture

control RNA –
Detection
Extract Separation sequencing
of DE
RNA of m-RNA and
challenged genes
analysis
 Variant (SNP) Detection
• Genome sequencing facilitates the identification of genomic variation within
different individuals by sequencing and comparing the data of individual genomes
with reference genomes.
• After successful alignment to a reference genome, SNPs are identified(heritable
variations in animal genome)
• Recent advances in NGS of DNA have enabled the systematic identification of
CNVs at a higher resolution and sensitivity
• SNP’s can be used to
 Analyze genetic diversity
 Characterization of genetic population structure
 Genetic mapping, QTL mapping
 Marker Assisted Selection and Breeding
 Detection of genetic diversity among populations of a species has important
effect on adaptive potential of a species

(Moore et al., 2011)

 Assembled domestic animal genomes
Bovine Womack, 2006
Water buffalo Michelizzi et al., 2010
Porcine Mote and Rothschild, 2006
Sheep Cockett, 2006
Horse Chowdhary and Raudsepp, 2006
Chicken Burt, 2006
Canine Galibert and Andre, 2006
Feline Murphy, 2006Pareek et al., 2011

Royal Bengal tiger genome sequenced

The high coverage genome sequencing and
identification of genome variants in Bengal tiger were
carried out by scientists from the Centre for Cellular
and Molecular Biology (CSIR-CCMB) and a
Hyderabad-based private company.
 Future challenges

 Defining variability in many human and animal genomes

 Analysis of vast production of sequencing database through advanced
bioinformatics tools
 Conclusions
• Recently, HT-NGS technologies emerged as a potential research tool for the
development of animal genomes research
• Major role in genetic improvement of animal health and productivity in near
future
• NGS can be used to study functional as well as comparative genomics
• Provided insight into epigenomic study
• Helpful in providing information about evolution