Recent Advances in NGS Technologies

1. “Recent Advances in
NGS Technologies
Copenhagenomics:
June 14, 2012

2. BCM-HGSC Sequencer Fleet
Life Tech SOLiDTM 4
Life Tech 3730 system Life Tech Ion Torrent PGM
Roche 454 FLX system Pacific Biosciences
Illumina GAIIx & Hiseq2000 2

3. Big Science Projects
• 1,000 Genomes Project
– Discovery of rare (1%) SNVs & SVs in normal genomes
• The Cancer Genome Atlas
– Discovery of sequence variants in major cancers
• Personal Genome Project
– Discovery of sequence variants associated with medical
information
• Human Microbiome Project
– Study of communities of mixed microbes within human niches
• The Exome Project
– Discovery of sequence variants in protein coding regions
• Pharmacogenomics Research Network
– Discovery of sequence variants involving drug-gene interactions
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4. NGS Applications
• Human biology
– Genotype – phenotype interactions
• Pharmacogenomics
– Drug – gene interactions
• Diagnostics
– Actionable variants
• Forensics
– Linking suspects to a crime scene
• Human Microbiome
– Study of microbe communities
• Many more than time to cover…
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5. Capture Sequencing at BCM-HGSC
Library Automation
Decrease Reagent cost
Decrease Labor cost
Increase capture production
Multiplex Sequence Capture
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6. HGSC Capture Projects
May 2008 to present
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7. Diagnostic Exome Sequencing
Joint effort between BCM’s HGSC and BCM’s
Medical Genetics Laboratories (MGL) to provide
exome sequencing with clinical interpretation 7

8. HIV forensics
State of Louisiana
v. Richard J.
Schmidt Metzker et al. (2002)
PNAS 99: 14292-14297
Patient → Trahan
State of Washington v.
Anthony Eugene
Whitfield
Whitfield → 5 partners
Scaduto et al. (2010)
PNAS 107: 21242-21247
State of Texas v.
Philippe Padieu
Padieu → 6 partners
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9. Direction of transmission
(source → recipient)
Providing evidence for the direction of transmission would
further strengthen the a priori hypothesis.
Genetic bottleneck during transmission
•Paraphyly: Evidence for direction of transmission
Study design:
•identities of case subjects were blinded to investigators
•case sample handling were separated both temporally and
spatially to eliminate the possibility of cross contamination
•case allegations were multiple transmissions from a single
source
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10. HIV genes: pol and env
RT (1-221 aa) gp120 (c2-v5)
Methods involved were:
•Fractionation of PBMCs
•Isolation of genomic DNA
•PCR and cloning
•Sanger sequencing
•Multiple sequence alignments
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11. Texas case: pol tree
CC01 exhibited a
paraphyletic relationship
to all CC case sequences
•Bayesian posterior
probabilities (1.00)
•ML bootstrapping proportions
(0.98)
Red circle represents the
most recent common
ancestor of sequences
from CC01
Scaduto et al. (2010) PNAS 107: 21242-21247
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12. Texas case: env tree
CC01 exhibited a
paraphyletic relationship to
all CC case sequences but
CC05
•Bayesian posterior probabilities
(1.00)
•ML bootstrapping proportions
(1.00)
Red circle represents
the most recent
common ancestor of
sequences from CC01
Scaduto et al. (2010) PNAS 107: 21242-21247
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13. NGS in HIV forensics
Development of the ‘pathogen toolkit’
Long-range PCR Clone analysis: EcoRI
10kb
5kb
4kb
3kb
Large insert cloning
NIJ grant: 2011-DN-BX-K534
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14. NGS in HIV forensics
Development of the ‘pathogen toolkit’
Case sample 01 Case sample 02 so forth
Molecular Fragment, add Molecular Fragment, add
clone 01 forward adaptor with MC01, clone 01 forward adaptor with MC01,
reverse adaptor with CS01 reverse adaptor with CS02
… …
… …
… …
Molecular Fragment, add Molecular Fragment, add
clone 20 forward adaptor with MC20, clone 20 forward adaptor with MC20,
reverse adaptor with CS01 reverse adaptor with CS02
Pool libraries,
then clonally amplify &
sequence by NGS technologies
NIJ grant: 2011-DN-BX-K534
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15. Emerging NGS Technologies
Is there room for new NGS
Technologies?
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16. Different NGS Strategies
• Single nucleotide addition
– Pyrosequencing and bioluminescence detection
– Natural nucleotide and H+ ion detection
• Reversible terminators
– Clonally amplified templates with 4-color detection
– Single molecule templates with 1-color detection
• Non-cleavable SBL
– Clonally amplified templates with 4-color detection
• Cleavable SBL
– Clonally amplified templates with 4-color detection
• Real-time
– Single molecule templates with 4-color detection
• Nanopores
– Single molecules translocated through small holes
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17. LaserGen
Lightning Terminators™
What differentiates LaserGen from other NGS technologies?
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18. Lightning Terminators™
H Fluor H Fluor
N N
Properties: O O
OMe OMe
• 3’-OH unblocked O 2N
NH 2
O2 N
O
t -Bu O t-Bu O
reversible terminators N
N
N
NH
N N NH 2
HO O O O HO O O O
• Fast incorporation
P P P O P P P O
O O O O O O O O O O O O
OH OH
kinetics LT-dA LT-dG
• Fast cleavage kinetics H
N
Fluor H
N
Fluor
O O
• High fidelity → OMe OMe
high accuracy O2 N NH 2 O 2N O
t-Bu O N t -Bu O NH
• Single-base HO
P
O
P
O
P
O
O
N O
HO
P P
O O
P
O
O
N O
termination O O O O O O
OH
O O O O O O
OH
LT-dC LT-dU
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19. Fast Incorporation Kinetics
k pol KD k pol/K D Selection
Nucleotide -1
(s ) (μM) (μM-1 s-1) (analog/TTP)
TTP 170 ± 4 73 ± 3 2.3 1.0
HOMedUTP 250 ± 11 33 ± 7 7.6 3.3
dU.V 37 ± 6 15 ± 2 2.5 1.1
dU.VI 36 ± 7 12 ± 1 3.0 1.3
• Fast incorporation rates
• Lower KD gives kinetic advantage over natural dNTPs
• As efficient as natural dNTPs
Gardner et al. (2012) Nucleic Acids Res., published May 8, 2012
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20. High Fidelity
Mutant
10
100004
Molecular tuning: polymerases "C"
Wild-type selectivity
"G"
•Key structural site drop fidelity "T"
identified 10
10003
)0
5
o
i C
I
t
a d
•Increasing size = R
y
t
i
v
i
t
e
h
c
t
a
c M
/
better specificity e
l
e
S
e
5 102
0
C 100
I
d
d
i e
t h
c
o
e t
l
c a
u m
s
N i
M
(
101
10
O NO2
CH 3
HN O CHCH3
3
H 3C CH 3
CH
3
O N
HO O O O
P P P O 100
1
O O O O O O
HOMedUTP HOMedUTP
HOMedUTP dU.I
Cpd dU.I dU.II
Cpd dU.II dU.III
Cpd dU.III dU.IV
Cpd dU.IV dU.V
Cpd dU.V
OH Vent(exo-) Therminator
Litosh et al. (2011) Nucleic Acids Res., 39:e39
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21. High Fidelity
Nucleotide selectivity
Template k pol KD k pol/K D k pol/K D (Corr.) IC50 (MisM) /
Nucleotide
base -1
(s ) (μM) (μM s )
-1 -1 / k pol/K D IC50 (Corr.)
a (MisM)
TTP C 72 ± 1 150 ± 8 0.48 4.9 11
G 55 ± 2 340 ± 50 0.16 14 22
T 97 ± 4 290 ± 19 0.33 7.0 44
a -3
dU.V C 0.045 ± 0.035 12 ± 2 3.8 x 10 630 1300
-3
G 0.030 ± 0.002 25 ± 1 1.2 x 10 2000 740
-3
T 0.053 ± 0.011 45 ± 4 1.2 x 10 2000 850
b -3
dU.VI C 0.063 ± 0.020 13 ± 3 4.8 x 10 620 590
-3
G 0.048 ± 0.016 44 ± 6 1.1 x 10 2800 400
-3
T 0.035 ± 0.010 34 ± 10 1.0 x 10 2900 540
Gardner et al. (2012) Nucleic Acids Res., published May 8, 2012
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22. Current progress
Status: UV
source
•E. coli genome
sequenced
•Pilot phase for mixed
culture studies
•Pathogen detection Microfluidic
flowcell
paper in preparation
Objective
Spectral
filters
Excitation
source
Digital
camera Mirror
Hertzog et al. (2011) BioOptics World DoD contract: W81XWH-12-C-0061
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23. Acknowledgements
LaserGen, Inc. BCM-HGSC NHGRI grants
Megan Hersh Priyanka R21 HG002443
David Hertzog Kshatriya R41 HG003072
Weidong Wu Huyen Dinh R01 HG003573
Hong Li Donna R43 HG003443
Brian Stupi Muzny R21 HG004757
Jinchun Wang Eric
Sidney Morris NEB
Boerwinkle NIJ grant
Cyril Chen Andy
Richard A. 2011-DN-BX-K534
Peng Chen Gardner
Gibbs
Michael Paras Bill DoD contract
U of Texas
Mimi Healy Jack W81XWH-12-C-0061
David
Hillis
LSU
Jeremy
Brown
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