2. DNA SEQUENCING
DNA Sequencing is Figuring out the order of DNA
nucleotides, or bases (A T G C ), in a genome that
make up an organism’s DNA.
F. Sangar
Sangar
Sequencing
3. History of DNA sequencing
1953
Discovery of the structure of the
DNA double helix
1972
Development of Recombinant
DNA technology,.
1977
The first complete DNA genome
to be sequenced is that of
Bacteriophage φX174 &
Frederick Sanger publishes
"DNA sequencing with chain-
terminating inhibitors“
1984
Medical Research Council
scientists decipher the complete
DNA sequence of the Epstein-
Barr virus, 170 kb.
1987
Applied Biosystems markets first
automated sequencing machine,
the model ABI 370.
1990
The U.S. National Institutes of
Health (NIH) begins large-scale
sequencing trials on M.
capricolum, E. coli
Caenorhabditis elegans and S.
cerevisiae
1995
Craig Venter Hamilton Smith
and colleagues publish the 1st
complete genome of bacterium
H. influenzae (whole-genome
shotgun sequencing.)
1996
Pål Nyrén and his student
Mostafa Ronaghi at the Royal
Institute of Technology in
Stockholm publish their method
of Pyrosequencing
1998
Phil Green and Brent Ewing
of the University of
Washington publish "phred”
for sequencer data analysis.
2001
A draft sequence of the
human genome is published.
2004
454 Life Sciences markets a
parallelized version of
Pyrosequencing.
2006
Era of Next Generation
Sequencing- 454
Sequencing, Illumina etc.
4. ERA OF SEQUENCING
1st Generation sequencing
• Sequence many identical molecules
• Sequencing in large gels or capillary tubing limits
scale
Sangar Chain Termination
( 1977 )
Maxam- Gilbert Sequencing
(1977)
ABI PRISM 377
5. 5
Intro to NGS, 11.30.2016
1st Generation Sequencing
• Sequence many identical
molecules
• Sequencing in large gels or
capillary tubing limits scale
2nd Generation Sequencing
• Sequence millions of clonally
amplified molecules per run
• Using a reversible, stepwise
sequencing chemistry
• Immobilized on a surface
ERA OF SEQUENCING
QIAGEN GeneReader
Life Technologies/Applied
Biosystems; SOLID 5500
Illumina MiSeq
Roche / 454
Pyrosequencer
6. NEXT GENERATION SEQUENCING
High throughput DNA Sequencing Technique.
Employs Micro and Nanotechnologies
Reduce sample size.
Low Reagent cost
Less Time
Massive Parallel Sequencing
Sequence thousands of sequences at once.
Produce enormous amount of data .
7. NGS WORKFLOW
Clonal Amplification by
Bridge PCR
Sequencing-by-ligation
(SOLiD Platform )
Clonal Amplification by
Emulsion PCR
Pyrosequencing
(454 Sequencing)
Sequencing-by-synthesis
( Solexa Technology)
Sample Extraction , DNA fragmentation and invitro adapter ligation
8. NGS WORKFLOW
1. Create DNA fragments
2. Add platform-specific adapter sequences to every fragment.
Adapter
ligation
point
Adapter
molecule
Adapter molecules : Bind library to a flowcell or bead; Add sequence primer
binding sites & Add barcodes for multiplexing.
Adapter molecule
bound to DNA .
10. Cluster Amplification:
Bridge PCR
DNA fragments are flanked with adaptors (library)
A solid surface is coated with primers complementary
to the two adaptor sequences
Isothermal amplification, with one end of each “bridge”
tethered to the surface
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
Fragments with adaptors (the library) are PCR amplified within a water drop in oil.
One PCR primer is attached to the surface of a bead.
DNA molecules are synthesized on the beads in the water droplet. Each bead bears
clonal DNA originated from a single DNA fragment
Beads (with attached DNA) are then deposited into the wells of sequencing chips –
one well, one bead
12. Sequencing & Imaging Technologies:
Chain Reversible Termination
Sequencing by Cyclic Reversible Termination (CRT): CRT uses
reversible terminators in a cyclic method that comprises nucleotide
incorporation, fluorescence imaging and cleavage. 100-150bp reads are
used.
13. Sequencing & Imaging Technologies:
Sequencing by Ligation
Sequencing by Ligation (SBL) uses the enzyme DNA ligase to identify the nucleotide present
at a given position in a DNA sequence.
14. Sequencing & Imaging Technologies:
Pyrosequencing
Pyrosequencing: non-electrophoretic, bioluminescence method that measures the
release of inorganic pyrophosphate by proportionally converting it into visible light using a
series of enzymatic reaction
Nucleotide incorporation generates light seen as a peak
in the Pyrogram trace
15. Sequencing & Imaging Technologies:
Single Molecule-Real Time Sequencing
Single Molecule- Real Time (SMRT) is a parallelized single molecule DNA
sequencing method. A single DNA polymerase enzyme is affixed at the bottom of a ZMW
with a single molecule of DNA as a template
DNA
Polymerase
ZMW
(Zero Mode
Waveguide DNA)
16. NGS Technologies Overview
NGS differs in template preparation, sequencing and imaging, and data analysis
Commercially available technologies:
Illumina/Solexa
Roche/454
Helicos BioSciences
Life/APG – SOLiD system
Pacific Biosciences
Ion Torrent technology
17. Solid-phase amplification 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
ILLUMINA/SOLEXA SEQUENCING
Run time: 1–10 days
Produces: 2–1000 Gb of sequence
Read length: 2 x 50 bp – 2 x 250 bp
(paired-end)
Cost: $0.05–$0.40/Mb
Bridge PCR Clustal Amplification
18. Applications
DNA sequencing
Gene Regulation Analysis
Sequencing-based Transcriptome Analysis
SNPs and SVs discovery
Cytogenetic Analysis
ChIP-sequencing
Small RNA discovery analysis
A whole human genome sequence was
determined in 8 weeks to an average
depth of ~ 40X, discovering ~ 4 new
million SNPs and ~400000 SVs (with an
accuracy <1% for both over-calls and
under-calls)
Over 1800
publications.
19. ROCHE/454 SEQUENCING
Sequence much longer reads by sequencing multiple reads at once by reading optical signals as
bases are added.
The DNA or RNA is fragmented into shorter reads up to 1kb.
Uses Emulsion PCR for Clustal Amplificication.
PYROSEQUENCING as sequencing approach.
20. Nucleotide incorporation generates light seen as a peak in the
Pyrogram trace .
All of the sequence reads we get from 454 will be different
lengths, because different numbers of these bases will be
added with each cycle.
Applications
Whole genome sequencing
Targeted resequencing
Sequencing-based Transcriptome Analysis
Metagenomics
Over 1300
publications...
21. ION TORRENT SEQUENCING
Ion torrent and ion 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.
Run time: 3 h; no termination or deprotection steps
Clustal Amplification- Emulsion PCR
Read length: 100–300 bp
Throughput determined by chip size : 10Mb – 5 Gb
Cost: $1–$20/Mb
The pH change, if any, is used to determine how many
bases (if any) were added with each cycle.
22. LIFE/APG/ABI- SOLiD SEQUENCING
AB SOLIDTM 3 System generates over 20 gigabases & 400 M tags per run .
Library Preparation
Emulsion PCR/ Bead Enrichment
Bead deposition
Sequencing by Ligation
2. Chemical crosslinking to
an amino-coated glass
surface
23. SANGERS Vs. NGS
Features Sanger NGS
Sequencing
Samples
Clones, PCR DNA Libraries
Preparation Steps Few, Sequencing reactions clean
up
Many, Complex
procedures
Data Collection Samples in plates :
96, 384
Samples on slides
1-16+
Data 1 Read/ Sample Thousands & Millions of
Reads/ Samples.
25. ADVANTAGES OF NGS
Sanger Sequencing NGS Sequencing
No invivo cloning, Transformation,
Colony picking
High degree of Parallelism then
Capillary Sequencing
Low Reagent Cost
Reduced Sample Size
Less Time
28. SUMMARY
Next Generation Sequencing has changed the way we
carry out molecular biology and genomic studies.
It has allowed us to sequence and annotate genomes at
a faster rate.
It has allowed us to study , variation, expression and
DNA binding at a genome – wide level.
29. REFERENCES
Elaine R. Mardis (2008) the impact of next-generation sequencing
technology on genetics. Cell vol.24 No.3,133-14.
Elaine R. Mardis (2009): Next-Generation Sequencing Methods. Annu.
Rev. Genomics hum genet. 9:387-402
Jorge S Reis-Filho (2010): Next-Generation Sequencing, Breast Cancer
Research 2010, 11(Suppl 3)
Some websites –
https://www.ncbi.nlm.nih.gov/pubmed
