Next Generation Sequencing of HLA: Challenges and Opportunities in the era of Precision Medicine. Dr. Paul Keown, 2016

Transcription

1 Next Generation Sequencing of HLA: Challenges and Opportunities in the era of Precision Medicine Dr. Paul Keown, 2016

2 Statement of Conflict & Collaboration Therapeutics collaborations Novartis, Roche, Astellas, Shire Diagnostics collaborations Roche, Luminex, ThermoFisher, Illumina, Pacific Biosciences, HTG, Omixon, ImmuCor University of British Columbia Patents on diagnostics in kidney and heart transplantation Corporate governance Syreon corporation, Syreon Research Institute, digital informatics, therapeutic research and economic analysis 2

3 Genomics, HLA and precision medicine Origin and technology selection Development: from protein, to RNA, to DNA Evaluation and selection for HLA typing in Canada Cost implications and challenges Capital and operating costs Laboratory and computing requirements Considerations for laboratory workflow Training, validation and implementation Organization of workflow, throughput and reporting Opportunities for the future Immunological monitoring throughout the graft course Genetics of immune disorders 3

4 History of Genome Sequencing Source: U.S. Department of Energy Office of Science, Systems Biology for Energy and the Environment, Human Genome Project Information Base URL: 4

5 Landmarks in gene sequencing 1958: Protein Sequencing 1968: RNA Sequencing 1980: DNA Sequencing 5

6 Landmarks in transplantation 1990: Organ transplant 1990: HSC transplant Dr. Joseph Murray, Harvard Dr. E. Donnal Thomas, Seattle 6

7 NGS: 2 nd generation sequencing systems Solexa: MPS Illumina MiSeq Roche 454 Life Technologies PGM 7

8 NGS: 3 nd single molecule systems 8

9 Canadian Blood Services (CBS) 9

10 Canadian Blood Services (CBS) 10

11 Canadian consensus conference 11

12 NGS platforms 12

13 Commercial HLA NGS methods 13

14 Canadian consensus conference: Report 1. All methods good, individual benefits / limitations 2. Selected a first method on basis of simplicity / cost 3. Will review all advances every 6 months & report 14

15 G3 NGS platforms 15

16 Genomics, HLA and precision medicine Origin and technology selection Development: from protein, to RNA, to DNA Evaluation and selection for HLA typing in Canada Cost implications and challenges Capital and operating costs Laboratory and computing requirements Considerations for laboratory workflow Training, validation and implementation Organization of workflow, throughput and reporting Opportunities for the future Immunological monitoring throughout the graft course Genetics of immune disorders 16

17 Costs of implementing NGS Capital equipment $ K Operating cost $150 $5-10K Evaluation Laboratory Costs: $SS Technologist training $SS $20K Validation and accreditation $SS Computer & connectivity 17

18 Capital equipment and automation DNA quantitation DNA qualitation DNA amplification Library size selection Sequencing Library quantitation DNA extraction DNA quantitation DNA qualitation Pre-PCR liquid handler DNA amplification Sequencing Library quantitation Library size selection Post-PCR liquid handler 18

19 Considerations in implementing NGS 19

20 Capital costs for NGS *Based on figures from four US Labs, rounded to $1,000 20

21 Costs (CAD) Costs (CAD) Operating (materials) costs for NGS 16, , ,000 10,000 8,000 6,000 4,000 Auxillary Sequencing Library Prep DNA quantitation per patient 11 loci per patient, per locus 2, per run of 24 per run of 48 per run of 96 0 per run of 24per run of 48per run of 96 21

22 Laboratory flow and space requirements Process flow Data analysis Upstream integration Sample collection and DNA extraction DNA sep Library preparation workflows Sample batching Liquid handling automation Data processing and analysis Data transfer automation Analysis automation Biobank Cytometry Core Post-PCR Thermo-cyclers Genomics Vortex Centrifuge Core Library prep. Sequencer Long-term data storage Approval workflow and tracking LIMS integration Export to NMDP standard Import directly into LIMS Molecular Core Accession Pre-PCR Thermocyclers Centrifuge 22

23 Laboratory and space requirements Before After 23

24 Computerization & connectivity Internal networking External networking 1. HSC network 2. SOT network 3. CBS network 24

25 Genomics, HLA and precision medicine Origin and technology selection Development: from protein, to RNA, to DNA Evaluation and selection for HLA typing in Canada Cost implications and challenges Capital and operating costs Laboratory and computing requirements Considerations for laboratory workflow Training, validation and implementation Organization of workflow, throughput and reporting Opportunities for the future Immunological monitoring throughout the graft course Genetics of immune disorders 25

26 Staff training and recruitment Directors, Technical supervisors Autoimmune core Histocompatibility core Technologist training Cytometry program for NGS core Month 1: theory and procedures Month 2: observation & training Month 3: supervised sequencing 6 Month 4: independent sequencing Month 5: review & certification 26

27 Staff training and recruitment Directors, Technical supervisors Autoimmune core Histocompatibility core Cytometry core

28 An integrated genomics training program 28

29 Clinical workflow: steps and times Day 1 Day 2 Day 3 Day 4 Day hr Accession Sequence hr DNA separation *Library prep. Analysis *Report hr Longrange PCR Sequence hr 29

30 Clinical workflow: steps and times Day 1 Day 2 Day 3 Day 4 Day hr Accession hr DNA separation Library prep. Sequence hr Longrange PCR Report hr Sequence 30

31 Bench time for library preparation Step Process Total time Hands-on Start time 1. Generate HLA amplicons 8 hrs 90 mins Day 3.00 pm 2. PCR cleanup 30 mins 30 mins Day 8.00 am 3. Amplicon normalization 1 hr 60 mins Begin Day Library prep 8.30 am Vendor 0 4. Pooling and library preparation 4 hrs 60 mins Day 9.30 am Vendor 1 5. Library size selection 45 mins 10 mins Day Begin 1.30 pm Vendor 2 6. Final quantification 1.5 hr 15 mins Day 2.30 pm Vendor 3 7. Sequencer loading 20 mins 20 mins Day 4.00 pm 2.5 same day Vendor 4 Total 16+ hrs < 5 hrs Hours Library prep next day 31

32 Process time for Sanger sequencing and NGS Sanger NGS Day O/N1 Target generation Target generation Day O/N1 Sequencing Library preparation Day 2 Day 2 Data analysis Clonal amplification Sequencing Sequencing Day 3 Day 3 Data analysis Data analysis Data 32

33 Validation and quality testing Summarized on pages 37 to 39 of ASHI Standards Parallel testing ASHI Validation Minimum 50 samples, minimum 3 runs Run to run variation Tech to tech variation Comparable run sizes to routine Same kind of samples (buccals, blood etc) Blind parallel testing Minimum 20 samples Quality Assurance Evaluate potential allele dropouts Document/validate preparation process for samples Including compliance with vendor specifications Monitor fidelity of barcoding methods Rotate control samples with different barcode sequences Instrument performance measures From internal control samples and/or vendor supplied material 33

34 HLA typing: laboratory process flow Samples PCR Typing Informatics Reporting HCT HCT teams HCT SOT SOT teams SOT AID AID teams AID Accession Review 34

35 Process for HLA typing: HCT SSO / SSP 6 loci SSO / SSP 2-6 loci T Patient T SSO / SSP 2 loci SSO / SSP 6 loci HCT Team A T Familial donor T R HCT Team SSO / SSP 6 loci SSO / SSP 6 loci T Unrelated donor T 35

36 Process for HLA typing: SOT SSO / SSP 4 loci Ab SSO / SSP 1-7 loci T Patient T SSO / SSP 4 loci Ab SSO / SSP 1-7 loci SOT Team A T Familial donor T R SOT Team SSO / SSP 4-11 loci Ab SSO / SSP 1-7 loci T Unrelated donor T 36

37 Process for NGS HLA typing: all organs HCT Team 2 samples NGX 6-11 Loci HCT Team A T R SOT Team SOT Team 37

38 Streamlined histocompatibility testing HLA-A SSO HLA-B SSO HLA-C SSO HLA-DRBI SSO HLA-DRB3,4,5 SSO HLA-DQA1/B1 SSO HLA-DPA1/B2 SSO HLA-A1-68 SSP HLA-B5-58 SSP HLA-Cw1-18 SSP HLA-DRB SSP HLA-DRB3,4,5 SSP HLA-DQA1/B1 SSP HLA-DPA1/B1 SSP NGS PAST FUTURE 38

39 Streamlined histocompatibility testing 96 samples x 4 sequencing primers 384 reactions (Sanger) 96 samples 1 reaction (NGS) NGS PAST FUTURE 39

40 Genomics, HLA and precision medicine Origin and technology selection Development: from protein, to RNA, to DNA Evaluation and selection for HLA typing in Canada Cost implications and challenges Capital and operating costs Laboratory and computing requirements Considerations for laboratory workflow Training, validation and implementation Organization of workflow, throughput and reporting Opportunities for the future Immunological monitoring throughout the graft course Genetics of immune disorders 40

41 The era of precision medicine 41

42 Disease association testing 42

43 Combinatorial effects in immune disease HLA-B27 spectrum B*27:05 B*27:09 Endoplasmic reticulum aminopeptidase 1 1 Sorrentino: 2 Alvarez-Navarro: 3 Colbert: Mol Immunol

44 Organ Function (%) Monitoring the continuum of disease Baseline Risk Disease Presence Disease Progression Restored Organ Function Earlier Intervention Improved Organ Function End-stage Markers of Organ Failure Recurrent Native Disease/Transplant Organ Failure Time (years) Transplantation, Assist Devices Biomarker panel opportunity Intervention point 44

45 Principal pathways control gene expression c-myc SP1 Blue wavy icons: generic binding proteins, yellow arrows: generic enzymes, green arrows: regulators. Blue dots: under-represented, Red dots: over-represented. The complete legend can be found at: 45

46 Immunity and inflammation in uremia A. Transcripts for many key cytokines are elevated in chronic renal failure, HD and PD (many peaking in PD), but expression levels return towards normal after transplantation B. Transcripts for many key chemokines and Toll receptors are suppressed in chronic renal failure, HD and PD (many reaching a nadir in HD and PD), but expression levels return towards normal after transplantation 46

47 Functional genomics and RNA-seq 47

48 Summary and conclusions NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training Start-up and capital costs may be high, but operating costs are low and decrease further with higher throughput Individual 2 nd generation assays and platforms have inherent limitations that require special care and expertise Technology is advancing rapidly and novel 3 rd generation systems hold enormous promise in accuracy and cost NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course 48