Next-generation sequencing and its application

1. Presented by:
Babu Lal Saini
Ph.D.
NGS and Its Application In
Livestock
ANIMAL GENETICS DIVISION
ICAR-INDIAN VETERINARY RESEARCH INSTITUTE

2. Outline
• Timeline of NGS platform
• Introduction
• First generation sequencing
• NGS work flow
• NGS Platforms
• Application of NGS
• Future challenges
• Conclusion

3. Timeline of DNA sequencing technologies and platforms

4. 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)

5. Next generation sequencing platforms

6. 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

7. 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

9. NGS Workflow
Sample
• Source of sample (DNA, RNA)
• Qualify and quantify samples
Library
• Prepare platform specific library
• Qualify and quantify library
Sequenc
ing
• Perform sequencing run reaction on NGS platform
Analysis
• Application specific data analysis pipeline
Grada et al.;2013

10. 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

11. 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

12. 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
(Metzker et al., 2010)
Light signal recorded by camera

13. 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

14. 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
http://res.illumina.com/documents/products/techsp
(Guo et al., 2008)

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

16. 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

17. Ligation and Imaging…Ligation chemistry
Mardis;2013

18. Ligation and Imaging…Ligation chemistry
Mardis;2013

19. Data analysis
Mardis;2013

20. 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

21. 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)

22. Pacific Biosciences(SMRT™)
The SMRT - considered as TGS
• Zero-mode waveguides (ZMW)
– Heart of the technology
– Detect world’s smallest light
volume
• 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)
DNA polymerase
ZMW

23. 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)

24. Comparisons sequencing platforms
Platform Amplificati
on method
Sequencing
chemistry
Signal
detection
method
Run time Advantage Disadvantage Accuracy
Sanger
sequencing
Vector
based
Chain
termination
Long read Expensive
Impractical for
larger genome
seq
99.9%
454gsflx
(Roche)
Emulsion
PCR
Pyrosequenc
ing
Light 23h Long read Expensive
Homopolymer
error
99.9%
Illumina( hi
seq, mi seq)
Bridge PCR Sequencing
by synthesis
Light 2 days Hts
Expensive
Short read
Long run time
98%
AB solid Emulsion
PCR
Sequencing
by ligation
Light 8 days Low cost / base Short read
Long run time
99.9%
Ion torrent Emulsion
PCR
Sequencing
by synthesis
Ph 3h Short run time
Less expensive
Longer read
lengths
Homopolymer
error
99.9%
Helicos No PCR TSMS 1gb/h No PCR 100%
Pacbio No PCR
SMRT
SMRT Light 20 min Longest read
Fast
No PCR
Low yield at
high accuracy
Expensive
100%
http://www.molgen.mpg.de/899148/ows2013

25. METAGENO
MICS
NEXT GENERATION SEQUENCING
GENOMICS
TRANSCRI
PTOMICS
EPIGENOM
ICS
Whole genome
seq
Exome seq
De novo seq
Targeted seq
RNA- seq
Targeted RNA
seq
Nc RNA seq
ChIP-Seq
Methyl-Seq
Ribosome
profiling
Microbiome
Seq
Applications of NGS

26. Three NGS levels: Which sequencing level should we
choose?

27. CELLS ISOLATED(PBM’C)
CONTROL
INFECTED/CHALL
ENGED
RNA SEQ OF
CONTROL
RNA Seq OF
INFECTED
READS READS
GMAP
MAPPED READS
RNA-SEQ DATA ANALYSIS USING CUFFLINKS
PACKAGE
(cufflink, cuffmerge, cuffdiff)
DETECTION OF DIFFERENTIALLY EXPRESSED
GENES
GENE EXPRESSION PROFILING THROUGH
RNA-seq
IDENTIFICATION OF GENES
UP OR DOWN REGULATED

28. SAGE (Serial Analysis of Gene Expression)
Velculescu V.E.(1995),Science 270:484-487

29. 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

30. 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

31. 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)

32. • Uncovering the potential
cause of the disappearance
of the honeybee
• To detect the cause of an
infectious disease
Cox-Foster,2007

33. 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

34. Genomic prediction in livestock using NGS
• NGS- explore relationship between genetic and phenotypic diversity with
high resolution
https://doi.org/10.12972/jabng.2017

35. challenged
control
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
Extract
RNA
Separation
of m-RNA
RNA –
sequencing
and
analysis
Detection
of DE
genes

36. • 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
Variant (SNP) Detection
(Moore et al., 2011)

37. 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.

38. Future challenges
 Defining variability in many human and animal genomes
 Analysis of vast production of sequencing database through advanced
bioinformatics tools

39. 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