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Clinical Applications of Next Generation Sequencing
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Clinical Applications of Next Generation Sequencing

Clinical Applications of Next Generation Sequencing

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  • $1.2 MOver 150 different tests
  • Baits: (biotinylated RNA oligos)
  • One lane 200M reads (base pair)One flow cell = 1.6 B readsMultiplex 4 samples to one lane ~50M reads
  • 100-200 million clusters
  • 175 days

Clinical Applications of Next Generation Sequencing Presentation Transcript

  • 1. CLINICAL APPLLICATIONS OF NEXT GENERATION SEQUENCING Bharat Thyagarajan Department of Laboratory Medicine and Pathology University of Minnesota
  • 2. MOLECULAR DIAGNOTSTICS LABORATORY • The Molecular Diagnostics Laboratory (MDL) processes around 20,000 specimens annually • Major testing categories – Infectious disease testing: HPV – Bone marrow engraftment analyses – Hematological malignancies • Translocations, quantitative BCR-ABL, JAK2/FLT3/NPM1/CEBPA. T and B cell gene rearrangements etc. – Solid tumor malignancies • Microsatellite instability, KRAS, BRAF etc. – Inherited disorders • Factor II, V mutations, sequencing, Southern blot etc.
  • 3. CURRENT MOLECULAR TESTING SCHEME FOR ONCOLOGY DNA/RNA EXTRACTED FROM SUBMITTED TISSUE MOLECULAR DIAGNOSTICS CLINICIANS ORDER INDIVIDUAL GENETIC TESTS CYTOGENETICS ONE MUTATION = ONE TEST FISH KARYOTYPE ARRAY CGH SEPARATE MOLECULAR PATHOLOGY REPORT SEPARATE CYTOGENETICS REPORTS
  • 4. LUNG CANCER • As recently as a decade ago stage IV lung cancer had an universally poor prognosis of < 12 months irrespective of chemotherapeutic regimen • At present, lung cancer with EGFR mutations have a mean survival of > 2 years • Initial results of targeted therapy for other genetic alterations (e.g. ALK, ROS translocations) have shown promise • Thus lung cancer is now considered a prototype for genetically tailored cancer therapy
  • 5. CURRENT GENETIC TESTING FOR LUNG CANCER • Molecular Diagnostics – EGFR mutation analysis • Wide range of genetic alterations including point mutations in various exons (18-21) and deletions in exon 19 • Cytogenetics – ALK-EML4 translocations • Commonly detected using an ALK break-apart probe
  • 6. MUTATIONAL PROFILE IN LUNG CANCER Kris, et al ASCO 2011 Erlotinib/Gefitinib Crizotinib MEK1/2 inhibitors?? Dabrafenib ROS1 Crizotinib
  • 7. CURRENT MOLECULAR TESTING SCHEME FOR INHERITED DISEASE DNA EXTRACTED FROM BLOOD MOLECULAR DIAGNOSTICS CLINICIANS ORDER INDIVIDUAL GENETIC TESTS CYTOGENETICS ONE GENE = ONE TEST FISH KARYOTYPE ARRAY CGH EACH TEST IS A SEPARATE MOLECULAR PATHOLOGY REPORT SEPARATE CYTOGENETICS REPORTS
  • 8. DISTRIBUTION OF MSI vs. MSS COLON CANCERS
  • 9. TESTING FOR LYNCH SYNDROME
  • 10. CURRENT TESTING ALGORITHM PATIENT WITH COLORECTAL CANCER MEETS AC/BG DOES NOT MEET AC/BG IHC NO FURTHER MSI TESTING NEGATIVE POSITIVE (LOSS OF MLH1, PMS2) MOLECULAR MSI TESTING BRAF MUTATION /hMLH1 METHYLATION NEGATIVE POSITIVE POSITIVE NEGATIVE NO FURTHER GENETIC TESTING REQURIED GENETIC TEST FOR MLH1 , MSH2, PMS2 GENES
  • 11. LIMITATIONS OF CURRENT TESTING PARADIGM • INHERITED DISORDERS – Comprehensive genetic testing for several syndromes frequently involve simultaneous testing for several genes – Increasing demand for detection of point mutations and structural genetic alterations within tested genes • CANCER DIAGNOSTICS – Comprehensive prognostic and predictive testing in near future will involve testing at least a few dozen genes – Various types of genetic alterations (point mutations, translocations etc.) will need to be evaluated simultaneously – Limited amount of sample available will be available for testing
  • 12. PROPOSED SOLUTION • NEXT GENERATION SEQUENCING technology was specifically designed to simultaneously evaluate variation in several genes • This technology can also be used to detect different types of genetic alterations • TYPES OF SEQUENCERS – HiSeq 2000/2500 – Desktop sequencers: MiSeq/IonTorrent
  • 13. TECHNOLOGY: NEXT GENERATION SEQUENCING
  • 14. WHOLE GENOME VS. TARGETED CAPTURE $5,000/sample
  • 15. MAJOR STEPS OF NGS • DNA library preparation • Target enrichment • Cluster generation & sequencing (Illumina HiSeq 2000) • Bioinformatics analysis of sequence data • Data interpretation
  • 16. Genomic DNA fragment 150-600bp in length The size of the fragment is called the “insert size” Adaptors Universal PCR primers Bar code (multiplex) Sequence that hybridizes to flow cell Paired-end read- 50-150bp of DNA are sequenced at each end fragment. If the insert size is 500bp, these reads should map ~500bp apart LIBRARY PREPARATION Library = fragments of DNA that have been prepared for amplification and sequencing
  • 17. SEQUENCE ENRICHMENT OPTIONS Multiplex PCR Microfluidics PCR
  • 18. PCR VS. SEQUENCE CAPTURE
  • 19. SEQUENCE CAPTURE • Up to 80% enrichment for the targeted DNA • 120 bp “baits” bind to DNA and magnetic beads • Unbound DNA is discarded • Baits are digested
  • 20. METHODOLOGIES FOR PERFORMING PCR BASED ENRICHMENT • Uniplex PCR: 1 reaction = 1 amplicon • Multiplex PCR: 1 reaction = 10-50 amplicons • Droplet PCR: 1 reaction = 4,000 amplicons • Microfluidics PCR – Multiplex 10 PCR/well – Can simultaneously amplify 480 amplicons
  • 21. CLUSTER GENERATION AND SEQUENCING • Oligonucleotides attached to flow cell hybridize to the adaptors • Individual DNA library fragments are immobilized
  • 22. • Starting DNA template concentration is crucial to avoid overcrowding of clusters • Each unique DNA molecule undergoes “bridge amplification” CLUSTER GENERATION AND SEQUENCING
  • 23. • Simultaneous generation of millions of clusters (“polonies”) • One cluster: – Derives from a single parent DNA molecule – Made up of ~1000 identical copies – Unique – Physically isolated from other clusters CLUSTER GENERATION AND SEQUENCING
  • 24. SEQUENCING BY SYNTHESIS • All clusters are sequenced in parallel, one base at a time • Fluorescently tagged nucleotides compete for next space • Fluorescent tag blocks addition of more than 1 nucleotide per round • Each round – Addition of one base – Laser excitation -> fluorescence – One “base” read from each cluster – Removal of fluorescent tag A G C T T TA T A G C T
  • 25. SEQUENCING BY SYNTHESIS
  • 26. BIOINFORMATICS ANALYSIS 3 days→5 hr 2 days→10 hr < 1 hr ~ 2 hrs 2 days→5 hr 1 day / ~ 2hrs
  • 27. INHERITED DISEASES VS. ONCOLOGY INHERITED DISEASES ONCOLOGY NUMBER OF GENES Range: 1-150 Range: 1-50 MUTATION DISTRIBUTION Across entire gene Hotspot mutations/deletions /translocations MINIMUM COVERAGE 20X ~250X – 1000X STRUCTURAL VARIATION Existing methods work well Sensitivity depends on tumor percentage TISSUE TYPES Blood Blood, Fresh frozen, FFPE, cytology INPUT DNA 3 µg 5 ng – 1µg TURNAROUND TIMES 4-6 weeks 5-7 days COST Range: $1000 - $10,000 Range: $400 - $1000
  • 28. TESTING SCHEME FOR INHERITED DISEASES SAMPLE PREPARATION TARGET CAPTURE SEQUENCING SMALL PANELS RAPID TURNAROUND TIMES (5-7 DAYS) LARGE PANELS EXOME SEQUENCING SLOW TURNAROUND TIMES (8-10 WEEKS)
  • 29. INHERITED DISEASES:NGS TESTING NEUROLOGY 223 GENES (39%) 31 DISEASES METABOLISM 14 GENES (2%) 10 DISEASES HEMATOLOGY 10 GENES (2%) 6 CONDITIONS HEARING LOSS 80 GENES (14%) 12 DISEASES CONNECTIVE TISSUE 15 GENES (3%) 6 DISEASES EYE 104 GENES (18%) 13 DISEASES BMT 33 GENES (6%) 5 DISEASES FAMILIAL CANCER 34 GENES (6%) 15 DISEASES DEVELOPMENTAL 51 GENES (9%) 9 DISEASES CARDIAC 4 GENES (1%) 4 DISEASES 0% 0% 0%
  • 30. NGS EXPERIENCE AT MDL • Have offered NGS testing for over 130 Mendelian disorders since August 2012 – We have tested 300 samples – We have detected mutations in approximately 30% of all samples tested – Mutation detection rate is dependent on clinical diagnosis • Mutation identified in 80% of inherited thrombophilias • Mutation identified in 25% of ataxias • No mutations identified in disorders of sexual development > 10 1 GENE 6-10 GENES 2-5 GENES
  • 31. GENOMIC REGIONS THAT ARE PROBLEMATIC FOR NGS 52 UNIQUE DISCREPANCIES IN CODING REGION OF DNA GC RICH REGIONS : 18 DISCREPANCIES (35%) POLYMORPHIC REPEAT REGIONS: 7 DISCREPANCIES (13%) MISMAPPED LARGE INDELS : 10 DISCREPANCIES (19%) TRI-ALLELIC SNPS: 1 DISCREPANCY (2%) PSEUDOGENES/ HOMOLOGOUS REGIONS: 16 DISCREPANCIES (31%)
  • 32. TESTING SCHEME FOR ONCOLOGY DNA/RNA EXTRACTED FROM SUBMITTED TISSUE TARGETED MUTATION DETECTION SMALL INDELS NEXT GENERATION SEQUENCING TARGETED CNVs TRANSLOCATIONS GENE EXPRESSION FISH/RT-PCR GENOMIC CNV ALTERATIONS Array CGH SAMPLE PREP: MICROFLUIDIC PCR RAPID SEQUENCING: MiSeq HiSeq2500 INTEGRATED CLINICAL REPORTS
  • 33. ONCOLOGY:NGS TESTING • Lung Cancer Panel • Somatic mutation testing – KRAS (NRAS/HRAS) – EGFR – BRAF – PIK3CA – ERBB2 – MET – TP53 – AKT1 – MAP2K1 – EGFRvIII (RT-PCR assay) • Translocation – ALK (EML4-ALK, but other partners up to 20) – ROS (up to 7 partners) – KIF5B/RET – CCDC6/RET (aka RET/PTC1) • Amplification – EGFR • MET • MAPK1 (p42/ERK2) • FGFR1 • FGFR2 • Gastrointestinal Cancer Panel • Somatic mutation testing – EGFR – KRAS (HRAS/NRAS) – BRAF – PIK3CA – TP53 – ERBB2 – MET – KIT – PDGFRA – AKT1 – PTEN – APC • Amplification/Deletion – ERBB2 – IGF2 (11p15.5) – PTEN – MDM2 – EGFR (rare)
  • 34. DETECTION OF EGFR MUTATIONS • Targeted sequencing of exons 18-21 (visualizing 18-19) • Input DNA: 50 ng of DNA for two lung cancer specimens • Specimen 1 (exon 19 deletion) – 80% tumor • Specimen 2 (L747P mutation due to sequential T>C mutations) – 70% tumor
  • 35. EGFR EXON 19 DELETION SAMPLE WITH DELETION AVG COVERAGE: 1800X NORMAL SAMPLE: AVG COVERAGE: 12500X EXON 18 EXON 18 EXON 19 EXON 19
  • 36. EGFR L747P MUTATION NORMAL SPECIMEN L747P MUTATION: 59% MUTANT ALLELE 15000 X COVERAGE
  • 37. 5 ng DNA 34% MUTANT ALLELE 25 ng DNA 12% MUTANT ALLELE 50 ng DNA 27% MUTANT ALLELE BRAF V600E MUTATION: MINIMUM DNA INPUT
  • 38. TECHNICAL ISSUES WITH NGS IMPLEMENTATION • Bioinformatics methods for sequence alignment keep undergoing rapid improvements – Need to update bioinformatics pipeline at frequent intervals • Structural genetic variation: – Optimal algorithms for detection of copy number variation remain unclear • Several regions with inadequate coverage – Backup Sanger sequencing/alternative methodology necessary for several exons in the context of inherited disorders – Sensitivity to detect somatic mutations will not be the same in all the analyzed regions
  • 39. CLINICAL ISSUES WITH NGS IMPLEMENTATION • Interpretation of clinical significance of many variants is unclear – Communication of these results to the clinician is problematic – Often results in additional testing of family members to determine clinical significance of a particular variant • Incidental genetic findings need to reported and appropriate clinical follow up procedures need to be in place
  • 40. OTHER ISSUES WITH NGS IMPLEMENTATION • High upfront costs for test validation – Substantial reagent costs • High sequencing run costs – Need to batch samples to reduce assay costs • Need to offer a large test menu to increase sample volume – Limited ability to repeat samples • Robustness of assays need to be adequately validated
  • 41. FUTURE APPLICATIONS OF NGS gDNA library creation NGS assembly, annotation Assembled Genome • instrumentation/consumables • sequencing per se • library in, reads out DNA SeqCap DNA Targeted re-sequencing/Exome Patient sample PCR DNA Microbial profiling DNA Mate Pair Deletion detection Community Characterization Structural Variants Polymorphisms Mendelian disorders/HLA typing Tumor DNA PCR DNA Microfluidics PCR Somatic mutation detection
  • 42. ACKNOWLEDGEMENTS • FUNDING SOURCES – Institute for Translational Neuroscience – Biomedical Genomics Center • BIOMEDICAL GENOMICS CENTER – Kenneth Beckman, Karina Bunjer, Adam Hauge, Archana Deshpande • BIOINFORMATICS CORE FACILITIY – Kevin Silverstein, Getiria Onsongo, Jesse Erdmann • MOLECULAR DIAGNOSTICS LABORATORY – Matt Bower, Teresa Kemmer, Matt Schomaker, Sophia Yohe, Jon Wilson, Michael Spears, Andrew Nelson • FAIRVIEW – Klint Kjeldahl, Karin Libby