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“Recent Advances in
NGS Technologies

      Copenhagenomics:
          June 14, 2012
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
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
                                                                        3
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…


                                          4
Capture Sequencing at BCM-HGSC




                              Library Automation




Decrease Reagent cost
Decrease Labor cost
Increase capture production
                              Multiplex Sequence Capture
                                                           5
HGSC Capture Projects
May 2008 to present




                        6
Diagnostic Exome Sequencing




  Joint effort between BCM’s HGSC and BCM’s
  Medical Genetics Laboratories (MGL) to provide
  exome sequencing with clinical interpretation    7
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

                                                    8
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

                                                                9
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
                                             10
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
                                              11
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
                                              12
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
                                                                   13
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
                                                                                                   14
Emerging NGS Technologies


     Is there room for new NGS
     Technologies?




                                 15
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
                                                            16
LaserGen
Lightning Terminators™


 What differentiates LaserGen from other NGS technologies?




                                                             17
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


                                                                                                                                                           18
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
                                                                               19
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
                                                                                                                                                          20
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
                                                                                                                21
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
                                                                                       22
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
                                                         23

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Recent Advances in NGS Technologies, LaserGen & Baylor College of Medicine, Michael L. Metzker Copenhagenomics 2012

  • 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 3
  • 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… 4
  • 5. Capture Sequencing at BCM-HGSC Library Automation Decrease Reagent cost Decrease Labor cost Increase capture production Multiplex Sequence Capture 5
  • 6. HGSC Capture Projects May 2008 to present 6
  • 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 8
  • 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 9
  • 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 10
  • 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 11
  • 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 12
  • 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 13
  • 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 14
  • 15. Emerging NGS Technologies Is there room for new NGS Technologies? 15
  • 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 16
  • 17. LaserGen Lightning Terminators™ What differentiates LaserGen from other NGS technologies? 17
  • 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 18
  • 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 19
  • 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 20
  • 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 21
  • 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 22
  • 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 23