Is NGS for everyone? Is NGS for everyone? Factors to consider in selecting a platform and approach. Costs as incentive and barrier

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1 NEX-GENERAION SEQUENCING (NGS) WORKSHOP November 10-11, 2016 Embassy Suites by Hilton Dallas - DFW Airport South Is NGS for everyone? Lee Ann Baxter-Lowe University of Southern California Children s Hospital Los Angeles NEX-GENERAION SEQUENCING (NGS) WORKSHOP November 10-11, 2016 Embassy CONFLIC Suites by Hilton OF Dallas INERES - DFW Airport South Lee Ann Baxter-Lowe, Ph.D. Clinical Professor University of Southern California Los Angeles, CA USA I have no financial relationships with commercial interests to disclose. My presentation does not include discussion of off-label or investigational use of drugs. My presentation includes discussion of investigational laboratory tests. Is NGS for everyone? Factors to consider in selecting a platform and approach. Costs as incentive and barrier Strategies for integrating NGS into lab workflow 1

2 Is NGS for my lab? yping indications yping volume and turn-around-time Local environment Cost Clinical yping: 1, 2, 3, or 4 fields? BM oday: 2 field omorrow? 3-4 field Solid organ transplant esp defining DSA, virtual crossmatches oday: 1-2 field omorrow? 2-4 field Disease association and pharmacogenetics oday: 1-2 field omorrow? 3-4 field he next advance: HLA expression GvHD Non-relapse mortality Petersdorf et al Blood 2014 HLA-C expression levels define permissible mismatches in hematopoietic cell transplantation 2

3 More reasons for 3 or 4 fields Any indication where genetic factors affecting expression could be important Research (clinical trials, basic & translational) More information is better Competition for funding Expectations for being state-of-the art Reference lab Academia ransition to future? yping entire MHC Widespread use of NGS (ID, cancer) Is NGS for my lab? yping indications yping volume and turn-around-time Local environment NGS users in institution Access to equipment Laboratory staff Bioinformatics support Institutional support Alternative typing methods Costs Balancing cost and turn-around-time A Run frequency ime /run Cost Samples/run Approach 3

4 A: run frequency is critical factor Run Frequency ime/run A 2/week 3 days 3-5 working days 2/week 2 days 2-4 working days Weekly 3 days 3-10 working days Weekly 2 days 2-9 working days Every 2 weeks Every 2 weeks 3 days working days 2 days working days Balancing frequency (samples/run) and cost Samples per run Cost Calculating Reagent Costs per Sample Fixed Sample Prep Reagent Cost: Amplification & Library Prep Kit Cost ($) # ests per kit Variable Sequencing Reagent Cost: Sequencing Reagents Kit ($) # Samples per sequencing run Slide provided by Curt Lind, hermo Fisher 4

5 otal Reagent Cost per sample decreases in a decreasing amount Sample prep cost remains the same however sequencing cost decreases Slide provided by Curt Lind, hermo Fisher Is NGS for my lab? yping indications yping volume and turn-around-time Local environment Cost Equipment access In lab Purchase Lease Reagent purchase Shared equipment Institutional core laboratory Send to high volume sequencing facility 5

6 Laboratory staff Expertise with HLA/molecular biology Product and vendor Robust products minimize need Vendor support reduces lab requirements NGS eliminates need for expertise to resolve ambiguities New challenges (optional?) Non-coding polymorphism Predicting impact of novel sequences Hands on time NGS can be less labor intensive than Sanger seq + ambiguity resolution I and bioinformatics support System set-up Server Data Storage Management Bioinformatics Predominantly commercial products Advantages to attract institutional support Cost effectiveness Importance for Patient care Research Educational environment Compatibility with other areas of laboratory medicine 6

7 NGS is becoming mainstream Cancer diagnostics Infectious disease Genetic diseases Is NGS for my lab? yping indications yping volume and turn-around-time Local environment Cost Factors to consider in vendor selection Lab s resources Platform Performance characteristics Practical Support 7

8 Factors to consider in evaluating products Lab s resources for evaluating products Conferences (e.g., ASHI meetings) Evaluation options On-site vs off-site Lab s cost for evaluation of product How many vendor evaluations can be supported by lab? Factors to consider in typing approach Platform beyond performance Institutional availability Institutional compatibility (backup) Capacity/cost of the chip/flow cell Flexibility (reagent alternatives for platform) If purchasing Cost of purchase/lease and maintenance Reagents Factors to consider in vendor selection Performance characteristics Accuracy Failure rate Ambiguities Gene coverage 8

9 What is an acceptable failure rate? Run Frequency Acceptable Failure rate A 2/week Highest 3-5 working days Weekly Low 3-10 working days Every 2 weeks working days Gene coverage Gene coverage Whole Gene Coverage Exon 2 to Exon 4 Exon 1 + Exon 2 to Intron 5 NGSgo-AmpX amplification primer Amplified exon GENDX U UR UR UR UR R U UR UR UR UR R HLA-A (3.1 kb) HLA-B (3.4 kb) HLA-C (3.4 kb) HLA-DRB1 ( kb) HLA-DQB1 ( kb) HLA-DPB1 (5.0 & 5.7 kb) HLA-DPA1 (4.7 kb) HLA-DQA1 ( kb) HLA-DRB3 (3.8 kb) HLA-DRB4 (0.4 & 1.3 kb) HLA-DRB5 (4.0 kb) 9

10 Gene coverage Phasing best with long amplicons Allele 1 Allele 2 A G C Allele 3 Allele 4 A G C No phasing with short amplicons if intervening sequence is the identical in both alleles A G Phasing possible with long amplicons Every approach has advantages Phasing Base call accuracy PCR efficiency Fragmented DNA Amplicon Length Long Short Best Best Best Best Long Short 10

11 Practical factors to consider Packaging Amenable to lab s run volume Flexibility for selecting loci Cost/sample Software User friendliness Features Quality metrics Ease of use/robust ime/run Platform constraints Automation Factors to consider in vendor selection Support echnical Bioinformatics Validation Sales he bells and whistles. Packaging Amenable to lab s run volume Flexibility for selecting loci Cost/sample Software User friendliness Features Quality metrics Ease of use/robust ime/run Platform constraints Automation 11

12 Fundamental NGS metrics Coverage Depth (number of times a base call is made at a given position) Uniformity Quality Scores Phred-like quality scores for each base call Generated by platform-specific algorithms Assuring the quality of next-generation sequencing in clinical laboratory practice Gargis et al Nature Biotech 2013 Quality Statistics: Depth of Coverage EU: O, ROW: O NGSengine Quality values (Phred-like scores) Historically developed to assess Sanger sequencing accuracy Used multivariate lookup tables Accurate across sequencing chemistires and instruments Algorithm for QV for NGS are system-specific All QV scores logarithmically related to probability of base calling error Q = -10 log 10 P Q Value Probability of Error Accuracy 10 1 in 10 90% 20 1 in % 30 1 in 1, % 40 1 in 10, % 50 1 in 100, % Sanger NGS 12

13 Quality score diminishes with read length Duke et al International Journal of Immunogenetics, 42: , 27 JUN 2015 owards allele level human leucocyte antigens genotyping assessing two next generation sequencing platforms: Ion orrent Personal Genome Machine and Illumina MiSeq PCR Artifact (PCR crossover, chimeric PCR) Primer extension, but partial length Denature Anneal Partial length product becomes primer for another allele or locus Extension Hybrid molecule Allele imbalance PCR efficiency is influenced by DNA sequence Numerous factors can contribute to differences in amplification efficiency Primer mismatch Denaturation efficiency GC content GC clamp Sequences that can disrupt polymerase binding 13

14 Quality Statistics: Percentage most frequent base call versus rest Homozygous 2 alleles ~50% Noise 40 Phasing Allele 1 Allele 2 Allele 3 Allele 4 A A G C G C Reads Phased A G Not Phased A If exceeds length of all reads, phasing will not be possible Accuracy Long reads Fragment length Duke et al International Journal of Immunogenetics, 42: , 27 JUN 2015 owards allele level human leucocyte antigens genotyping assessing two next generation sequencing platforms: Ion orrent Personal Genome Machine and Illumina MiSeq 14

15 Software filters reads, criteria vary Low quality value Short reads PCR crossover No alignment Multiple possible alignments Read usage 15

16 he raffic Light System Example of metrics for HLA typing Fragment size Read length Read quality Read count Noise ratio Exon spot noise ratio Non-exon spot noise ratio Exon allele imbalance Non-exon allele imbalance PCR crossover artifact ratio Crossmapping (intergenic ambiguity) Ambiguous layout (intragenic ambiguity) Continuous consensus Fully phased consensus Consensus coverage exon minimum depth Consensus coverage non-exon minimum depth Genotype available Exon mismatch count Non-exon mismatch count Quality Control 16

17 Data analysis with NGSengine Mappability Read depth # possible genotypes Phasing regions Read length and typing result Mismatches EU: O, ROW: O Review Window Data Review All content 2016 Immucor, Inc. Review Window 2 Identification of Correct Allele Overall Coverage Plot Correct vs. Incorrect Alleles Central Coverage Plot Correct vs. Incorrect Alleles 51 All content 2016 Immucor, Inc. 17

18 Genotype Summary 52 Proprietary & Confidential Genotype Summary Health (Quality) Metrics: Uniformity of Coverage Allele Balance Full Key Exon Coverage (<20X) Exon Mismatches (>0) 53 Proprietary & Confidential Genotype Summary Allele 2 Allele 1 Max Read Depth Coverage Plot (Min Read Depth >200) 54 Proprietary & Confidential 18

19 Assign 2.0 Coverage View 55 Research Use Only. Not for use in diagnostic procedures. Scisco Genetics GeMS-HLA Software Summary All systems automate assignments and allow user to drill down. Quality metrics are important for Acceptance of automated assignment Identify typings for manual interpretation rouble shooting Criteria for acceptance should be validated by the laboratory Many programs available Most quality metrics determined by every program Presentation variable Important to be knowledgeable about software 19

20 Next Generation Sequencing Workshop November 10-11, 2016 Embassy Suites DFW Airport South 20