September, 2016 Agenda Introduction Overview of NGS Accreditation - - PowerPoint PPT Presentation
NGS Accreditation with ASHI Standards, Plus Omixon Update Presenters: Dr. Peter Meintjes, CCO, Omixon Tim Hague, CEO, Omixon Guest Speakers: Dr. Lee Ann Baxter-Lowe, CHLA Doreen Sese, Yale University Dr. Zahra Kashi, Kashi Labs Prof.
September, 2016
Presenters:
Tim Hague, CEO, Omixon Guest Speakers:
Doreen Sese, Yale University
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§ Introduction § Overview of NGS § Accreditation with ASHI Standards § Guest Speakers § Omixon Update
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§ Global molecular diagnostics company
– Budapest, Headquarters, Software R&D – Cambridge, MA, Operations and Manufacturing
§ World leader in HLA Typing by NGS
– Best in class Assay – Best in class Software
§ Existing Products
– Holotype HLA (Assay + Software) – HLA Twin (Software) – HLA Explore (Software)
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“Recent technological advances, cumulatively called NGS, provide the first opportunity to fully characterize and fully phase the HLA genes” –
§ The Technique
– Long Range PCR Amplification – Clonal sequencing of Amplicons – Assembly of whole gene consensus sequences
§ The Goal
– Automated, high-throughput analysis – Unambiguous results – No reflexive testing
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§ Summarized on pages 37 to 39 § Parallel testing
– Minimum 50 samples, minimum 3 runs – Comparable run sizes to routine – Same kind of samples (buccals, blood etc)
§ Blind parallel testing
– Minimum 20 samples
§ Quality testing
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Quality Testing
§ 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
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Quality Testing
§ Analytic performance criteria
– Incorporating vendor specifications – Base quality, read length, average coverage, uniformity of coverage
§ Independently validate software program
– Ensure genotyping algorithms are appropriate for sequencing strategy and sequencing error modalities
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§ Dr. Zahra Kashi – Kashi Labs § Dr. Lee Ann Baxter-Lowe - CHLA § Doreen Sese – Yale University
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§ Zahra Kashi, Ph.D, HCLD § Kashi Clinical Laboratories, Inc. § Portland, Oregon
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§ None
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§ Highly accurate § Almost no ambiguities § Almost full gene sequences § Single method § Is it the GOLD standard?
– NHGRI discrepancies – Failure of SBT to detect a second allele in a sample
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§ Before you invest:
– Is HLA your only application?
§ Instrument § Ongoing costs
– Which manufacturer?
§ Workflow and processes § Throughput and TAT
– Validation approach
§ Ongoing quality
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§ Initial Capital expenditure § Cost of “add-ons”
– Ancillary equipment
§ Cost of ongoing operation
– Consumables – Reagents – Labor (user’s ability to do other things) – Data management and storage
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§ Staff time for upfront setup (NGS)
– Amplicon PCR -> Pooling & cleanup -> Fragmentation / end-repair -> Adaptor ligation / Barcoding -> pooling and purification -> Quantitation
§ Staff time with Sanger - analysis and ambiguity
resolution
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§ Analytical sensitivity and specificity § Accuracy § Precision § Limit of detection
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1-3 pm The NGS Era Is Now Eric Weimer Jennifer Zhang
3:30 – 5:30 Is NGS for Everyone? Lee Ann Baxter-Lowe Paul Keown
approach.
5:30-7:30 pm Networking Entire faculty
Embassy Suites complimentary beverages and appetizers
Thursday afternoon, November 10
ASHI Workshop on
Next-Generation Sequencing (NGS)
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6:00 - 8:00 Networking breakfast Complementary breakfast in Embassy Suites Atrium 8:00 - 8:45 Challenges in Data Interpretation Marcelo Fernandez- Vina
8:45 – 9:30 Validation Requirements Eric Weimer
10:00 - noon Implementation insights
with
ASHI Workshop on
Next-Generation Sequencing (NGS)
Friday morning, November 11
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12:30 – 3:30 The NGS Marketplace
Box lunch 3:45 – 4:30 NGS beyond HLA Typing Marcelo Fernandez- Vina Lee Ann Baxter-Lowe
4:30 – 5:30 Faculty
5:30-7:30 pm Networking
Embassy Suites complimentary beverages and appetizers
ASHI Workshop on
Friday afternoon, November 11
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Member Registration: $250 before Oct 10 $300 on-site Non-Member Registration: $350 before Oct 10 $400 on-site Embassy Suites DFW S $129/night*
*single/double room, excluding applicable state and local taxes
ASHI Workshop on Next-Generation Sequencing (NGS) November 10-11, 2016
Embassy Suites Dallas - DFW Airport South
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EUR, AFA, API, HIS
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Variable Runs (n) Samples/Run (n) Typings (n) Samples/run 2 4 8 2 6 12 2 8 16 2 12 24 HLA types 1 8 8 Intra-assay 3 4 12 Inter-assay 2 4 8 Inter-tech (training) 3 4 12 Blind 1 4 4 Blind 2 8 16 Total 21 120
A SOLID ORGAN TRANSPLANT LAB’S JOURNEY
Doreen Sese, CHS (ABHI) Histocompatibility & Immune Evaluation Laboratory Yale University, Department of Transplant Surgery September 29, 2016
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Ø Support living & deceased donor solid organ transplant
programs (kidney, liver, pancreas, heart)
Ø Eliminated serology typing in 2012 Ø Current Methods: SSO, SSP, RT-PCR, NGS Ø Approximately 150 HLA typings per month Ø 6 full time technologists + 1 part time technologist Ø Shared on call
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Ø Met with Omixon Spring 2014 Ø Implementation of NGS instrumentation, Fall
2014
Ø Evaluation of different NGS kits early 2015 Ø 200+ Parallel samples started Fall 2015 Ø 20 Blind samples January – February 2016
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Ø 200+ parallel samples Ø 50 blinds Ø QA, QC, & Quality Metrics Ø Instrument qc & maintenance plans Ø Tech Training/Competency Ø Inter-assay reproducibility Ø Intra-assay reproducibility Ø 2 batches of 96 samples (higher sample range) Ø 2 – 3 batches of 24 samples (lower sample
range)
Data Management System Plan
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Ø Changing ASHI Guidelines Ø Continual software updates Ø Kit modifications / troubleshooting Ø Training & Staffing Ø Lack of IMGT sequences, DPB1 Ø Learning curve for data analysis Ø Server implementation
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Omixon Illumina
Yale IT
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??
1 2 3 4 5
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Ø Histotrac limited to 2000 alleles per locus Ø Need to update allele dictionary on an ad hoc basis Ø No provision for documenting new alleles in database Ø Hospital LIS (EPIC) update needed (> 3 fields appear
as blanks)
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§ All A locus ambiguities due to A*02:01:01:02L,
except for 1 new allele which was concordant
§ DRB1*12:01 vs DRB1*12:10 § DPB1 ambiguities due to lack of IMGT
sequence, but all within G groups
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Ø QC of new kit lots / shipments Ø Software updates Ø Kit updates Ø Technologist training
25% (6/24) samples in each run are controls
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Ø 24/11 kit validation completed last week Ø 2 Beckman Coulter Biomek 4000 liquid
handlers (1 pre, 1 post) installation this week
Ø Validation of liquid handlers, next week Ø Stand alone method by the end of the year with
the exception of deceased donor typings
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§ Prof. Dimitri Monos
Tim Hague
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§ 11 locus Holotype HLA kit – DPA1 and DRB3/4/5 – 24 or 96 sample kits, can be split up to three times § 300 cycle chemistry (paired 150bp reads) – Faster sequencing (24 hours instead of 40) § Extra indexing plates – Dual 24 and 96 sample index plates, alternating – Up to 192 samples per MiSeq run § V2 protocol – Reduced hands-on time, reduced turn around time § V2 software – Advanced interpretation tools, improved clinical workflow
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§ Remove gels (Optional) – Saves 30 mins HoT, 60 mins TAT § Dilution Optimization – Saves 20 + 35 mins § Rapid ExoSAP-IT – Saves 0 + 55 mins § Qubit Quantitiation (Optional) – Saves 0 + 40 mins
§ Bead-based size-selection (Optional)
– Saves 0 + 40 mins § Total savings – 50 mins HoT, 3 hours 50 mins TAT – In combination with 300 cycle flow cell saves a whole day
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§ V1
– Library Prep 7.2 hours – Results on day 5
§ V2
– Library Prep 4.3 hours – Results on day 3
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§ Interpretation
– Amino acid calling and visualisation – Noise visualisation – Fragment visualisation – Key exon QC metrics – QC reorganisation
§ Accuracy
– Optimized DRB3/4/5 analysis, including Linkage Disequilibrium check – Polyploidy detection
§ Usability
– Clinical workflow improvements, bulk assignment/approval – Improved results summaries
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QC Re-organisation and Key Exon QC Metrics
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ASHI validation study at CHOP 253 samples Double blind alpha study 16 samples, 6 labs each Clinical routine 2500+ clinical samples at CHOP Product evaluation study 200 samples 99.9% accuracy 98.5% sensitivity 99.2% reproducibility Worldwide Early Access Program 2400+ samples, 24 labs
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§ More than 90 evaluators § More than 20 labs in clinical production in Europe and the US
– 3 ASHI accredited labs
§ National tender for French Blood Services (EFS), 25k+ samples
per year
§ Alp Sen Foundation in Turkey, 20k+ samples per year § Two NHS labs in the UK
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§ Holotype HLA - updates
– 576 samples per run on the NextSeq – V2 product in Q4 – Automation on multiple robots
§ Monotype HLA
– New Product – HLA-B and DQB/A only
§ HLA Explore
– New Product – RUO software, for big data and long read data
Peter Meintjes, PhD Chief Commercial Officer peter.meintjes@omixon.com Tim Hague Chief Executive Officer tim.hague@omixon.com Maggi Woronkowicz Director of Sales maggi.woronkowicz@omixon.com Faisal Mojadiddi Director of Sales faisal.mojadiddi@omixon.com
Visit us at Booth 415!
Efi Melista Head of Lab and Field Operations efi.melista@omixon.com