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Clinical applications of NGS


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Clinical applications of NGS

  1. 1. Clinical Applications of NGS - Genomics, NIFTY and Beyond Dr. Ying(Abby) LIU, MD PhD
  2. 2. The stories all began with these images!
  3. 3. Principle of High Throughput Sequencing 1. incorporation 1. Incorporation 1. Incorporation 2. Elute and scan 2. Elute and scan 3. Cleavage C C C CT T A A G G 3
  4. 4. BGI Brief• Largest genomics research center in the world – 137 Hiseq2000, 27 AB Solid, 1 Roche 454, 2 Miseq, 1 Ion Torrent, 30 AB 3730• Strong leader board and qualified employees – More than 4000 employees including 1500 bioinformaticians• Extraordinary super computer and cutting-edge cloud computing technique – 102T flops/ 10PB storage/ 20TB memory• Numerous high-quality publications on top academic Journals – Nature/ Science/ …
  5. 5. BGI Introduction• Founded in 1999, for 1% of HGP• Now the largest genomic organization in the world 9.1.1999 in London
  6. 6. Milestones of BGI BGI-Beijing, 1999 BGI-Americas, 2010 BGI-WH, 2010 BGI-HZ, 2001 BGI-SZ, 2007BGI-Europe, 2010 BGI-HK, 2009
  7. 7. The Only Gene Giant Covering 55 Countries (Science 2.2012) Copenhagen Kobe PhiladelphiaUCD Shenzhen SA Canberra
  8. 8. Partners3000+ Institutes/Hospitals,15000+ Collaborators
  9. 9. Selected Large-Scale Projects in BGI
  10. 10. Selected Top Publications of BGI
  11. 11. Discovery Based on Large Data 1977 1980 1996 1998 1998 2000 2001 2002 2004 2005 2006 2007 2008 2009 2010 2011 20011.Life periodic table can be summarized2.It will have laws as genetic central dogma3. It can be elaborated in mathematical language 15
  12. 12. Organization Structure
  13. 13. BGI’sGene Test Strategy for Hereditary Diseases Before Before Prenatal Newborn Marriage Pregnancy Screening/ Screening/Screening Screening Diagnosis Diagnosis Monogenic diseaseMonogenic disease Monogenic disease NIFTY Hearing impairmentHearing impairment Hearing impairment Monogenic disease Inherited metabolic Hearing impairment diseases
  14. 14. NIFTY- Non-Invasive Fetal Trisomy Test-BGI Experience
  15. 15. Screening tests Diagnostic tests• Serum biochemical test; • Karyotyping (G- ultrasound scan banding or FISH)• Non-invasive • Invasive• Affordable • Expensive• Less accurate • Highly accurate• Low detection rate • High detection rate• High false positive rate • Low false positive rate
  16. 16. A safe prenatal screening test for fetal chromosomal aneuploidy Screening tests Diagnostic tests• Serum biochemical test; • Karyotyping (G- ultrasound scan banding or FISH)• Non-invasive • Invasive• Affordable • Expensive• Less accurate • Highly accurate• Low detection rate • High detection rate• High false positive rate • Low false positive rate
  17. 17. What is NIFTY Test?Non-Invasive Fetal TrisomYA superior screening testAnalysis of fetal cell free DNA in maternal plasmaBased on NGS and bioinformaticsEvaluate the likelihood of fetal trisomy 21, 18, 13High detection rate and low false positive rate
  18. 18. Scientific Discovery Fundamental Features of Cell-Free Fetal DNA• Short fragments of 145-200bp, derived from placental trophocytes• 970 times greater than fetal cells DNA in maternal blood.• Detectable in maternal plasma from the 5th week of gestation.• Concentration increase as the gestation age grows• Disappears soon after childbirth
  19. 19. The Principle of NIFTY cell-free DNA 50bp Chr6 Chr18 ChrX AACGT------ Chr21 CGATA------- Bioinformatics Chr7 Sequencing & TAGGC------ Chr13 ATCGC------ Chr11 Alignment ChrY Normal Sequence Counting T 21Cov-chrN = Effective number of sequencing fragments on chr N of sample Bioinformatics Effective number of sequencing fragments on chr N of reference sequence AnalysisChrN t-Score Cov-Depth—Cov-Depth of reference sample =Of testing sample Standard deviation of reference sample log ( Possibility of normal) Report L-value= log (Possibility of abnormal) 26
  20. 20. The Statistic Model of NIFTY 100 cell-equivalent free DNA /ml plasma 95from mother, 5 from fetus Normal Pregnancy T21 PregnancyMother Chr 21: Fetus Chr 21: Mother Chr 21: Fetus Chr 21: 95X2=190 5X2=10 95X2=190 5X3=15 In maternal blood In maternal blood Total Chr 21=190+10=200 Total Chr 21=190+15=205 Statistics for Tscore and L score • GC correlation • Binary hypothesis statistics analysis
  21. 21. The Performance Comparison of Statistics-I Our approach with GC Standard z-score approach z-score approach with GC- correlation and binary bias removal hypothesis t-test Items Diagnostic (number of cases) Sensitivity (%) Specificity (%) Sensitivity (%) Specificity (%) Sensitivity (%) Specificity (%) Trisomy 13 detection (2 cases) 50% 99.89 100% 100% 100% 100% Autosomes Trisomy 18 detection(12 cases) 91.67% 100% 100% 99.89% 100% 99.96% Trisomy 21 detection (16 cases) 93.75% 100% 100% 100% 100% 100% XO detection (3 XO,1 not not not not XO/XX chimera) 75% 99.5% available available available available Heterosomes not not not not XXY detection (1 case) 100% 100% available available available available not not not not XXY detection (2 cases) 100% 100% available available available available Binary hypothesis not available not available availableNote: Data from 903 samples with full karyotypinginformation 28
  22. 22. The Performance Comparison of Statistics-IIComparison of BGI’s approach (green)with other approaches (orange and yellow)PCT/CN2011/001070NONINVASIVE DETECTION OF FETAL GENETIC ABNORMALITY
  23. 23. Clinical Validation (Phase I)Study Design and Aim: – In 3464 Samples 3464 singleton pregnancies – Validation of the NIFTY in predicting with high risk of T21, T18, T13 the fetal risk of trisomy 21, 18, and 13 in high risk population by a double Plasma DNA from maternal peripheral blood blind test – Evaluation of sensitivity and specificity NIFTY test of the NIFTY by comparing to karyotyping result (clinical gold Double blind standard) Karyotyping by AF, CVS, Cord Bioinformatics analysis blood – Provide supports for large-scale test in Calculate sensitivity and real clinical setting specificity
  24. 24. NIFTY Validation Results (Phase I) T21 T18 T13 NIFTY positive 189 64 10 Karyotyping positive 188 63 10 False positive No. 1* 1* 0 False Negative No. 0 0 0 Sensitivity 100.00% 100.00% 100.00% Specificity 99.97% 99.97% 100% False Positive Rate 0.03% 0.029% 0.00% False Negative Rate 0.00% 0.00% 0.00% Positive Predictive Rate 99.49% 98.44% 100.00%*caused by insufficient sequencing depth
  25. 25. Large-scale Clinical Test (Phase II) Qualified maternal blood samples (n=11184) • Gestational week from 9 – 28 weeks, averagely 20 weeks • Maternal age from 18 – 45 years, averagely 31 years • 4522 screening test high risk pregnancies • 2426 screening test low risk pregnancies • 2720 other high risk factors (AMA, abnormal NT, previous abnormal pregnancy, etc.) • 1387 screening not done 0.7% of all samples Unable to produce results (n=79; failed DNA extraction, library construction, or sequencing)99.3% of all samples Pregnant women with NIFTY results (n=11105) Positive (n=190) Negative (n=10915) Dan, S., et al., Prenatal Diagnosis, 2012: p. 1-8
  26. 26. NIFTY Results in 11105 Samples Positive Results Negative No. of Cases T21 T18 T13 Results NIFTY 11,105 140 42 0 10915 Karyotyping 182 + 2818 139 41 0 2818False Positive Rate 0.03% 0.03% n/aFalse Negative Rate 0% 0% n/a Detection Rate 100% 100% n/a Specificity 99.6% 99.6% n/a Dan, S., et al., Prenatal Diagnosis, 2012: p. 1-8
  27. 27. Dan, S., et al., Prenatal Diagnosis, 2012: p. 1-8 Real Clinical Setting in 11,105 Singleton PregnanciesMaternal age Median (yr) 31 Advanced maternal age (>=35 yr) 3,858(34.74%)18–24 yr (n, %) 918(8.27%) 25–29 yr (n, %) 3,439(30.97%) 30–34 yr (n, %) 2,890(26.02%) 35–39 yr (n, %) 3,188(28.71%) ≥40 yr (n, %) 670(6.03%)ParityNullipara (n) 6,053/11,105(54.51%)Gestational age at blood sampling Median (wk) 20 Range (wk) 9-28 9 to 12 week (n, %) 371(3.34%) 13 to 16 week (n, %) 1,963(17.68%) 17 to 20 week (n, %) 5,598(50.41%) 21 to 24 week (n, %) 2,665(24.00%) 25 to 28 week (n, %) 508(4.57%)History or family history of aneuploidies (n) 68/11,105(0.61%)Presence of sonographic markers of chr abnormalities (n) 317/11,105(2.85%)Had conventional Down syndrome screening tests Yes – Screening positive (n, %) 4,522/6,948(65.08%) Yes – Screening negative (n, %) 2,426/6,948(34.92%) No – with one or more of other risk factors* (n, %) 2,770/11,105(24.94%) No – without any risk factors* (n, %) 1,387/11,105(12.48%)
  28. 28. Current Clinical Pipeline AMA, Previous affected fetus, Recurrent miscarriage, Aneuploidy background Dan, S., et al., Prenatal Diagnosis, 2012: p. 1-8
  29. 29. NIFTY Clinical Pipeline >98% of invasive Advanced Maternal Age, procedures could be avoided Previous affected fetus, Recurrent miscarriage, Aneuploidy background Dan, S., et al., Prenatal Diagnosis, 2012: p. 1-8
  30. 30. Data Summary(Jan 2013 updated) Testing ResultsPhases Total cases T21 T18 T13Up-dated Multi-centered Clinical test of NIFTY 90847 724 253 65(including the data of Phase I& II validation)Subtotal for Phase I: Clinical Trial Study (2009-2010) 3464 189 64 10Detection Rate >99.9% >99.9% >99.9%False Positive Rate 0.0121% 0.0080% 0.0040%PPV 99.38% 98.67% 96.43%False Negative Rate 0% 0% 0%NPV 100% 100% 100%
  31. 31. Other Rare Aneuploidies-IIT9Sample ID: PDB11AJ00026Age: 41NIFTY: T9Karyotyping: T9
  32. 32. Other Rare Aneuploidies-IIIT16Sample ID:PDB11AJ00783Age: 30NIFTY: T16FISH: T16
  33. 33. Other Rare Aneuploidies-IVMosaic T21Sample ID: PDB12AO00267Age: 38NIFTY: T21Karyotyping: 47, XX, +21(88%)
  34. 34. Other Rare Aneuploidies-IVT21-T7-XXY complex placental mosaicSample ID: PDB12AL00732Age: 37NIFTY: T21-T7-XXY/XYKaryotyping: CVS T21-T7-XXY/XY; AF euploid
  35. 35. NIFTY is Suitable for Gestational age 12-24 weeks Recurrent AMA or highmiscarriage or risk IVF NIFTY Refuse to Inappropriate receive invasive for invasive test test
  36. 36. NIFTY Workflow Hospital Pre-test Blood Bar- Plasma Post-testcounselin preparation collection coding counseling g BGI Clinical Laboratories: 10 days Laboratory Bioinformatics Report testing analysis delivery
  37. 37. BGI SystemsSample Management Laboratory Effective lab space separation Unique Identification Progressively decreased pressure Sample Location Restricted traffic flow Storage Capability 46
  38. 38. Sample RequirementsBlood sampling Packing for delivery• 5-10ml in EDTA tube • Strong support in case of• Clearly labeled damage • Enough dry ice to keep frozenPlasma preparation Sample storage• Immediately extract • -20℃ for a week, -80℃ for plasma long term• Stored at 4℃, extract in 8 • Avoid room temperature hours and repeated thaw
  39. 39. NIFTY Genetic Testing ReportLow risk:-The fetus is unlikely to be T21, T18, or T13-No special medical procedure is recommended-Routine prenatal checks is suggestedHigh risk:-The fetus is likely to be T21, T18, or T13-Diagnostic procedure such as amniocentesis orcordocentesis is recommendedMore than 98% of cases
  40. 40. Other Rare AneuploidiesChromosomal number variations (CNVs) on otherautosomal chromosomesChromosomal number variations (CNVs) on sexchromosomesResults of other prenatal tests such as biochemical andultrasound tests should be consideredDiagnostic procedure such as amniocentesis/cordocentesisis suggested
  41. 41. BGI Papers 52
  42. 42. NIFTY SummarySA F E 5mlmaternal blood Sensitivity99.9%AC CU R A T E Specificity99.91%FA S T 10working daysEA R L Y from12thweek on 54
  43. 43. Monogenic Diseases Test-BGI Experience
  44. 44. Background• a single mutated gene, Mendelian pattern of inheritance Autosomal dominant Autosomal recessive• point mutation, frame shift, deletion, X-linkage dominant insertion X-linkage recessive Y-linkage• Autosomal dominant/recessive; X- linked dominant/recessive; Y-linked• >6700 monogenic diseases,10-50 more every year.• 1 in every 200 newborns• Genetic testing is the gold standard
  45. 45. Monogenic Disease Strategies at BGITarget region capture combined with NGSExome/whole genome resequencingSanger sequencing
  46. 46. Target Sequence Capture + NGS Target region PCR, capture, Sanger NGS sequencing
  47. 47. Mutation DatabasesdbSNP Hapmap 1000 genome Local genomefrequency frequency project database Normal persons SNV database Single-gene disease variants database 59
  48. 48. AdvantagesHigh throughput• Multiplex testingFull coverage• Simultaneously detect point mutation, minor insertion/deletion (<20bp), and large homozygous deletion/duplicationAccurate• Test region covers >95% target gene, sensitivity >99%Automated bioinformatics analysis• Fast identify mutation; completed disease mutation database and control genetic polymorphism database available
  49. 49. Monogenic Diseases Psychological health & behavior 9 diseases Visual system Brain & Nervous system 16 diseases 1 disease Ear, Nose & Throat 6 diseases Oral & dental 1 disease Immune system 6 diseases Congenital tumor Respiratory system 2 diseases Circulatory system 54 diseases Endocrine & Metabolic system Digestive system 7 diseases 8 diseases Multi-system Blood & lymphocyte system 6 diseases Urinary & reproductive system 5 diseases Musculoskeletal & 25 diseases Connective tissue system Skin, Hair & Nails 7 diseases160 common monogenic diseases across 16physiologicaland functional systems
  50. 50. List of Monogenic Diseases (160 diseases) Disease Gene and mutation INPP5E,TMEM216,TMEM67,OFD1,NPHP1,CC2D2A,CEP290,ARL13B,Joubert syndrome Disease AHI1,RPGRIP1L Gene and mutationSpastic Paraplegia, Autosomal Recessive and X-linked Alport Syndrome, X-Linked CYP7B1,SPG7,SPG11,ZFYVE26,SPG20,SPG21,L1CAM,PLP1,SLC16A2 COL4A3,COL4A4,COL4A5 DiseaseSpastic Paraplegia, Autosomal Dominant dominant Polycystic Kidney Disease, Autosomal PKD1,PKD2 Gene and mutation ATL1,SPAST,NIPA1,KIAA0196,KIF5A,HSPD1,BSCL2,REEP1,ZFYVE27 Polycystic KidneyIIDisease, Autosomal Recessive Citrullinemia, TypeMetachromatic Leukodystrophy PKHD1 ASS1,SLC25A13,ASL ARSA CA4,CRX,FSCN2,IMPDH1,NRL,PRPF3,PRPF31,PRPF8, ACADVL RetinitisDehydrogenase,Form Long-Chain, Deficiency Of Acyl-Coa Pigmentosa, VeryRefsum Disease, Infantileautosomal dominant PEX1,PEX2,PEX26 Acyl-Coa Dehydrogenase, Short-Chain, Deficiency OfArts syndrome PRPH2,RHO,ROM1,RP1,RP9 ACADS PRPS1 Acyl-Coa Dehydrogenase, Medium-Chain, Deficiency OfAgenesis Of The Corpus Callosum With Peripheral Neuropathy ABCA4,BEST1,CERKL,CNGA1,CNGB1,CRB1,EYS,IDH3B, ACADM SLC12A6 Argininosuccinic Aciduria KLHL7,LRAT,MERTK,NR2E3,PDE6A,PDE6B,PRCD,PROM1, ASLPeutz-Jeghers Syndrome autosomal recessive and X-link recessive Retinitis Pigmentosa, STK11 Argininemia PRPH2,RGR,RLBP1,RP2,RPE65,RPGR,SAG,SEMA4A, ARG1Adenomatous Polyposis Of The Colon APC Aspartylglucosaminuria SNRNP200,TOPORS,TULP1,USH2A,RHO,NRL AGAGilbert syndrome UGT1A1 Usher Syndrome to cystathionine beta-synthase deficiency Homocystinuria due USH2A,CDH23,CLRN1,GPR98 CBSCystic Fibrosis CFTR Waardenburg syndrome Tyrosinemia Type ITuberous Sclerosis EDNRB,MITF,PAX3 FAH TSC1,TSC2 Glutaric Acidemia I Osteogenesis Imperfecta GCDH COL1A1,COL1A2,CRTAPMarfan Syndrome FBN1 IsovalericAcidemia Osteopetrosis IVD CLCN7Bloom Syndrome BLM Fabry Disease Amyotrophic Lateral Sclerosis GLA SOD1,FUS,SETX,TARDBP,ANG,VAPB,FIG4,ALS2 ACTG1,CCDC50,COCH,DFNA5,DIAPH1,DSPP,EYA4,GJB2,GJB3,GJB6 Krabbe Disease GALCNonsyndromic Deafness,Duchenne Dominant (including mitochondiral Muscular Dystrophy, Autosomal Type/Becker Type DMD ,GRHL2,KCNQ4,MIR96,MYH14,MYH9,MYO1A,MYO6,MYO7A, Niemann-Pick Disease NPC1,NPC2,SMPD1deafness) Congenita, Autosomal Dominant /Amyotonia Congenita Myotonia POU4F3,SIX1,SLC17A8,TECTA,TMC1,WFS1,m.1494C>T,m.1555A>G, CLCN1 AGL,ALDOA,ENO3,G6PC,GAA,GYS1,LDHA,PYGM,PGM1, m.7445A>G,m.7510T>C,m.7472insC,m.7511T>C Glycogen Storage Disease Epiphyseal Dysplasia, Multiple,Autosomal Recessive SLC26A2 PHKG2,GBE1 Galactosemia Permanent Neonatal CDH23,DFNB31,DFNB59,ESPN,ESRRB,GJB2,GJB6,GRXCR1,HGF, GALT Diabetes Mellitus, GCK,KCNJ11,INS,ABCC8,PDX1,PTF1A LOXHD1,MYO15A,MYO3A,MYO6,MYO7A,OTOA,OTOF,PCDH15,RDXNonsyndromic Deafness,Type II Nongoitrous Hypothyroidism, Congenital, Recessive (including mitochondiral Mucopolysaccharidosis Autosomal TSHR,TSHB,NKX2-5 IDS ,STRC,TMIE,TMPRSS3,TRIOBP,TMC1,USH1C,SLC26A4,BSND, Phenylketonuria Dystrophica, Autosomal Dominantdeafness) Epidermolysis Bullosa PAH COL7A1 PTPRQ,LHFPL5,LRTOMT,SLC26A5,MARVELD2,CLDN14,TPRN,LHFPL5, congenital Dysplasia,cortical hyperplasia Ectodermal adrenal Hidrotic, Autosomal Dominant CYP21A2 GJB6 m.1494C>T,m.1555A>G,m.7445A>G,m.7510T>C,m.7472insC,m.7511T>C Maple Syrup Urine DiseaseDeafness, X-linked Recessive BCKDHA,BCKDHB,DBT FANCA,FANCB,FANCC,BRCA2,FANCD2,FANCE,FANCF,FANCG, POU3F4,PRPS1 Fanconi Anemia Wilson Disease ATP7B FANCI,BRIP1,FANCL,FANCM,PALB2,RAD51C,SLX4Ataxia-Telangiectasia ATMMultiple Endocrine Neoplasia RETNeurofibromatosis NF1,NF2Exostoses, Multiple EXT1,EXT2Hereditary Breast and Ovarian Cancer BRCA1,BRCA2
  51. 51. Workflow Pre/posttest Sample DNA Library counseling collection extraction construction Report Family Bioinformatic Sequencing delivery validation analysisTurnaround time: 35 working days
  52. 52. Data analysis• Sequencing quality assessment (depth, coverage)• Variation sites (dbSNP frequency, HapMap frequency, 1000 genome frequency, local database frequency)• Possible mutations (nucleotide change – amino acid change)• Validation & confirmation of pathogenic gene
  53. 53. Report Template
  54. 54. This Test is Recommended for …Clinics:• Patients with clear phenotypes• Patients with suspected disease• People with no phenotypes but have family history• Carrier screeningResearch:• Assisting diagnosis: provide genetic proof for uncertain phenotypes• Disease research: deeper understanding of genetics, human mutations
  55. 55. Monogenic Disease Gene Detection• 160 Monogenic diseases across 16 physiological and functional systems• Peripheral blood or genomic DNA• Turnaround time: 35 working days• Available at all life stages – life cycle solution Before Before Prenatal Neonatal marriage pregnancy
  56. 56. And Beyond…• Genetic testing – Thalassemia&hemopathy – HPV, HCV, HBV – Cancer – Metabolic Disorders – Hearing Impairment – Microdeletion&Microduplication – PGD – Arrhythmia – High resolution HLA typing – Exome Sequencing – Personal Genome – …
  57. 57. Clinically Affordable Soon $1000 genome will be true very soon
  58. 58. Recent Promotions• NIFTY (3.18.2013 - 3.29.2013) + FREE Test for Newborn Metabolic Disorders + FREE Test for Hearing Loss• HPV Testing (3.18.2013 - 4.18.2013)
  59. 59. Life Cycle Solution &Grand Scale
  60. 60. More or