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NGS
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
Analysis
• Application specific data analysis pipeline
Grada et al.;2013
Cluster Amplification: Bridge PCR
Used by Illumina
– Flow cell is coated with primers complementary to the two adaptor sequences
– Isothermal amplification
– 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
http://res.illumina.com/documents/products/techsp
Illumina Solexa Analyzer
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
• Procedure
1. Sample preparation
2. Emulsion amplification
Mardis;2013
Ligation and Imaging…Ligation chemistry
Mardis;2013
Data analysis
Mardis;2013
Ion Torrent PGM sequencer
• Sequencing platform does not uses optic signals
• Concept
• 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
• 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
Longer read
lengths
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
READS READS
GMAP
MAPPED READS
IDENTIFICATION OF GENES
UP OR DOWN REGULATED
SAGE (Serial Analysis of Gene Expression)
• Epigenetics – study of heritable gene expression that does not involve DNA
sequence.
– DNA methylation
• 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
https://doi.org/10.12972/jabng.2017
Selection of Disease Resistant Animals For Breeding
• 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
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