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. Dimitri Monos, CHOP September, 2016
Agenda § Introduction § Overview of NGS § Accreditation with ASHI Standards § Guest Speakers § Omixon Update www.omixon.com support@omixon.com
Corporate Statement § 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) www.omixon.com support@omixon.com
Omixon Website www.omixon.com support@omixon.com
Omixon Academy www.omixon.com support@omixon.com
My Holotype www.omixon.com support@omixon.com
Omixon on YouTube www.omixon.com support@omixon.com
Next Generation Sequencing “Recent technological advances, cumulatively called NGS, provide the first opportunity to fully characterize and fully phase the HLA genes” – Prof. Dimitri Monos, CHOP § 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 www.omixon.com support@omixon.com
ASHI Standards for NGS § 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 www.omixon.com support@omixon.com
ASHI Standards for NGS 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 www.omixon.com support@omixon.com
ASHI Standards for NGS 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 www.omixon.com support@omixon.com
Guest Speakers § Dr. Zahra Kashi – Kashi Labs § Dr. Lee Ann Baxter-Lowe - CHLA § Doreen Sese – Yale University www.omixon.com support@omixon.com
HLA Typing by NGS § Zahra Kashi, Ph.D, HCLD § Kashi Clinical Laboratories, Inc. § Portland, Oregon www.omixon.com support@omixon.com
Conflict of Interest § None www.omixon.com support@omixon.com
Next Generation Sequencing of HLA § 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 or noisy SBT trace www.omixon.com support@omixon.com
Implementing NGS § Before you invest: – Is HLA your only application? § Instrument § Ongoing costs – Which manufacturer? § Workflow and processes § Throughput and TAT – Validation approach § Ongoing quality www.omixon.com support@omixon.com
Budget § 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 www.omixon.com support@omixon.com
Workflow § Staff time for upfront setup (NGS) – Amplicon PCR -> Pooling & cleanup -> Fragmentation / end-repair -> Adaptor ligation / Barcoding -> pooling and purification -> Quantitation -> Sequencing § Staff time with Sanger - analysis and ambiguity resolution www.omixon.com support@omixon.com
Essential Performance Characteristics § Analytical sensitivity and specificity § Accuracy § Precision § Limit of detection www.omixon.com support@omixon.com
Learning Curve and Learning to Trust Data www.omixon.com support@omixon.com
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THANK YOU www.omixon.com support@omixon.com
ASHI Workshop on Next-Generation Sequencing (NGS) November 10-11, 2016 Embassy Suites Dallas - DFW Airport South www.omixon.com support@omixon.com
ASHI Workshop on Next-Generation Sequencing (NGS) Thursday afternoon, November 10 1-3 pm • Advantages of NGS-based typing The NGS Era Is Now • Current platforms/chemistries Eric Weimer Jennifer Zhang • Current typing systems • PCR options • Typing workflow 3:30 – 5:30 • Factors to consider in selecting a platform and Is NGS for Everyone? approach. Lee Ann Baxter-Lowe • Costs as incentive and barrier Paul Keown • Strategies for integrating NGS into lab workflow 5:30-7:30 pm • Informal discussions in lounge Networking Entire faculty • Vendors available in exhibit areas Embassy Suites complimentary beverages and appetizers www.omixon.com support@omixon.com
ASHI Workshop on Next-Generation Sequencing (NGS) Friday morning, November 11 6:00 - 8:00 Complementary breakfast in Embassy Suites Atrium Networking breakfast 8:00 - 8:45 • Problems Challenges in Data Interpretation • Rare alleles Marcelo Fernandez- Vina 8:45 – 9:30 • Requirements for validation Validation Requirements • Determining performance characteristics Eric Weimer • Establishing acceptance criteria 10:00 - noon • Faculty members will describe their experience Implementation with insights • Selecting methods • Integrating NGS into their lab workflow • Unexpected problems www.omixon.com support@omixon.com
ASHI Workshop on Next-Generation Sequencing (NGS) Friday afternoon, November 11 12:30 – 3:30 • Sequential vendor presentations The NGS Marketplace Box lunch 3:45 – 4:30 • MHC NGS beyond HLA Typing • NGS Projects in the 17 th International workshop Marcelo Fernandez- Vina • KIR Lee Ann Baxter-Lowe • Chimerism testing 4:30 – 5:30 • Panel discussion Faculty 5:30-7:30 pm • Informal discussions in lounge Networking Embassy Suites complimentary beverages and appetizers www.omixon.com support@omixon.com
ASHI Workshop on Next-Generation Sequencing (NGS) November 10-11, 2016 Embassy Suites Dallas - DFW Airport South 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 www.omixon.com support@omixon.com
Validation for NGS at CHLA • Designed for small lab with diverse patient population • 4-8 samples/week • Sample selection • For each locus, 90-100% of 10 most frequent alleles in EUR, AFA, API, HIS • Rare alleles • Allele combinations that challenge system • Homozygotes for most common types • Robust results for techs who perform many other assays • Validation plan revised several times due to • Changes in ASHI validation requirements • Software upgrades • New kit format www.omixon.com support@omixon.com
Validation plan if started October 2016 Variable Runs (n) Samples/Run Typings (n) (n) Samples/run 2 4 8 2 6 12 2 8 16 68 2 12 24 HLA types 1 8 8 Intra-assay 3 4 12 Inter-assay 2 4 8 32 Inter-tech 3 4 12 (training) Blind 1 4 4 20 Blind 2 8 16 Total 21 120 www.omixon.com support@omixon.com
FROM LOW RESOLUTION HLA TYPING TO NGS ACCREDITATION: A SOLID ORGAN TRANSPLANT LAB’S JOURNEY Doreen Sese, CHS (ABHI) Histocompatibility & Immune Evaluation Laboratory Yale University, Department of Transplant Surgery September 29, 2016
Laboratory Testing Background 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 www.omixon.com support@omixon.com
NGS Timeline 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 www.omixon.com support@omixon.com
Accreditation Plan (based on May 2015 Guidelines) 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) www.omixon.com support@omixon.com Data Management System Plan
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