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

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

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Recent Advances in NGS Technologies

Recent Advances in NGS Technologies

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

    • “Recent Advances inNGS Technologies Copenhagenomics: June 14, 2012
    • BCM-HGSC Sequencer Fleet Life Tech SOLiDTM 4Life 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 AutomationDecrease Reagent costDecrease Labor costIncrease capture production Multiplex Sequence Capture 5
    • HGSC Capture ProjectsMay 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 → TrahanState 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 wouldfurther strengthen the a priori hypothesis.Genetic bottleneck during transmission•Paraphyly: Evidence for direction of transmissionStudy design:•identities of case subjects were blinded to investigators•case sample handling were separated both temporally andspatially to eliminate the possibility of cross contamination•case allegations were multiple transmissions from a singlesource 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 themost recent commonancestor of sequencesfrom CC01Scaduto 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 CC01Scaduto et al. (2010) PNAS 107: 21242-21247 12
    • NGS in HIV forensicsDevelopment of the ‘pathogen toolkit’ Long-range PCR Clone analysis: EcoRI 10kb 5kb 4kb 3kb Large insert cloningNIJ grant: 2011-DN-BX-K534 13
    • NGS in HIV forensicsDevelopment 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 technologiesNIJ 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
    • LaserGenLightning Terminators™ What differentiates LaserGen from other NGS technologies? 17
    • Lightning Terminators™ H Fluor H Fluor N NProperties: 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 dNTPsGardner 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 NHO 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 -3dU.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 -3dU.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 progressStatus: 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 MirrorHertzog et al. (2011) BioOptics World DoD contract: W81XWH-12-C-0061 22
    • AcknowledgementsLaserGen, 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