NGS Accreditation with EFI Standards, Plus Omixon Update Presenters: Marcello Scala, Director of Sales EMEA, Omixon Tim Hague, CEO, Omixon Guest Speakers: Dr. Milena Ivanova, Bulgarian Bone Marrow Donor Registry Dr. Brendan Clarke, Leeds St. James University Hospital Dr. Mette Christiansen, Danish Bone Marrow Registry, Aarhus University May, 2016
Agenda § Introduction § Overview of NGS Accreditation with EFI 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
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
NGS workflow www.omixon.com support@omixon.com
EFI Accreditation for NGS § EFI has introduced new standards for accreditation using NGS techniques § Omixon’s Holotype HLA workflow and product provides full support for EFI accreditation § New standards include: – Documentation Guidelines – Nucleotide and Allele Assignment Standards – Bioinformatics Standards www.omixon.com support@omixon.com
Documentation Guidelines § Purification, sample tagging, normalization, pooling methods – Holotype HLA protocol, workbook, HLA Twin software § Steps must be taken to prevent creation of PCR artefacts, PCR artefacts must be documented – Optimized PCR conditions, QC metrics in software § Method of fragmentation must be specified, for each run the size of fragments must be documented and the selection must be specified – Documented in protocol, QC metrics and fragment distribution visualisation in software www.omixon.com support@omixon.com
Documentation Guidelines § Methods for enrichment strategies of multi gene panels must be defined. – Documented in Holotype HLA protocol § The conditions for the sequencing reaction must be documented and appropriate - including specificity, quantity and quality of templates; sequencing primers and sequencing reagents. – Documented in protocol, genotyping call in the software § Controls and procedures must be established to identify sample mix- up – Documented in workbook, any contamination will be detected by software www.omixon.com support@omixon.com
Allele Assignment § The criteria for acceptance of data must be established – 18 QC measures per locus in HLA Twin, 2 orthogonal algorithms § The signal to noise ratio must be sufficient to ensure reliable nucleotide assignments – 3 QC metrics for noise measurement, noise reduction algorithms § Interpretation, acceptance and/or rejection of sequences from regions which are difficult to resolve – Two orthogonal algorithms, QC measures, Genome Browser www.omixon.com support@omixon.com
Allele Assignment § Adequate depth of coverage thresholds – 2 QC metrics and thresholds for coverage § Determine the phase of alleles for the methods where phasing is possible – QC metric and 'phasing track' in Genome Browser www.omixon.com support@omixon.com
Bioinformatics § Sequencing metrics and QC parameters for optimal performance must be documented, specified and in range – 18 QC metrics per locus, with locus-specific thresholds § Each deviation from the standard operation procedure must be documented – All analysis parameters are logged and audited in the software § Detailed documentation and validation of the bioinformatics process supporting the analysis, interpretation and reporting results must be established – Omixon has already supported 75 labs through the evaluation process www.omixon.com support@omixon.com
Bioinformatics § Revalidation of bioinformatics processes must be performed after upgrading or changes of any affected components – Backward compatibility of the software, re-analysis may be required for IMGT/HLA database updates § Storage and back-up of data (input, raw data, intermediate and final data) must be defined in accordance with the national laws – Simple archive/storage mechanisms for Omixon results § The version of the bioinformatics process must be traceable for each sample analysed – Audited and logged www.omixon.com support@omixon.com
Bioinformatics § Periodic barcode rotation is required to detect contamination. If contamination is detected Standard L8.3 must be followed – Multiple plates (both 24 sample and 96 sample versions). Also contamination QC metrics in the software. § Algorithms for modification of raw sequence reads must be described in detail and validated (i.e. sequence trimming, quality filtering) – QC metrics and statistics visualisation for every locus/sample § Each sample processed must be traceable through the whole process including data analysis and reporting – Every sample, every allele assignment, every allele override, every lab manager approval is audited and reported www.omixon.com support@omixon.com
Guest Speakers § Dr. Milena Ivanova § Dr. Brendan Clarke § Dr. Mette Christiansen www.omixon.com support@omixon.com
HLA Typing Using Holotype HLA™ (Omixon) – Our Experience M. Ivanova Dept. of Clinical Immunology with Stem Cell Bank, University Hospital Alexandrovska, Medical University, Sofia, Bulgaria
The Evolving NGS Technology 2004-2015 www.omixon.com support@omixon.com
NGS Platforms Roche 454 Illumina IonTorrent SOLiD – PacBio RS 5500xl Amplification emPCR bridge PCR emPCR emPCR Non SMS NGS PS RTs Semiconductor Cleavable Real Time chemistry probe SBL Read length 400-650bp 150-600bp 200-400 110 3000 Output/run 0,65 Gb 1,5-500 Gb 0.1-12 Gb 155 Gb 0.5 Reads 10x6 1 1-2000 0,5-70 1410 0.03 Run time 10-20 h 10 h - 6 d 2.3 -7.3 h 8 d 2 h Errors Indel substitution Indel A-T bias Indel Error rate 1 0.1 1 <0.1 <1 www.omixon.com support@omixon.com
QUESTIONS???? § Research or clinical application § Goals – high thought or ultra high resolution § Application of HLA typing – solid organ transplantation, HSCT, Registry… § Number of samples § Time § Previous experience with sequencing technologies § Available platform and technology § NGS HLA typing is included in EFI Standards for Histocompatibility & Immunogenetics Testing v6.3 (Effective from Oct. 1. 2015) www.omixon.com support@omixon.com
Genetic test in Dept. of Clinical Immunology, University Hospital Alexandrovska EFI Accredited since 1997 Solid organ transplantation Other genetic markers: KIR, MBL, Cytokines, Diseases mtDNA, MPN mutations, PID HLA Typing Chimerism monitoring HSCT Cord blood bank Bone Marrow Donor Registry www.omixon.com support@omixon.com
Allele Level HLA Typing? Holotype HLA ™ X4 (Omixon) Initial evaluation on 16 samples, HLA typed by PCR-SBT using AlleleSeqR kits. Ambiguities were resolved by HARPs and PCR-SSP www.omixon.com support@omixon.com
Work Flow Holotype HLA ™ DNA preparation: hands on time - 35 min (35 min) HLA amplification: 35 min (7h 20) Amplicon quantification and normalization: 45 min (45min) Library preparation: 1h 10 (3h 15) Fragmentation; end repair; indexed adaptor ligation; library pooling and purification Library size selection (Pipin Prep): 15min (1h) Library quantification and MiSeq sequencing: 25 min (41h) Data analysis – Omixon HLA Twin v1.1.2 Hands on time 4h; Total time – 53h 30 www.omixon.com support@omixon.com
LR-PCR Amplification Strategy Full ¡gene ¡characteriza/on ¡ HLA-‑A, ¡B, ¡and ¡C ¡(Example ¡is ¡HLA-‑B) ¡ 5’ ¡UTR ¡ 1 ¡ 2 ¡ 3 ¡ 4 ¡ 5 ¡ 6 ¡ 7 ¡ 3’ ¡UTR ¡ HLA-‑DQB1 ¡ 5’ ¡UTR ¡ 1 ¡ 2 ¡ 3 ¡ 4 5 ¡ 6 ¡ 3’ ¡UTR ¡ Key ¡region ¡characteriza/on ¡ HLA-‑DRB1 ¡ 5’ ¡UTR ¡ 1 ¡ 2 ¡ 3 ¡ 4 5 6 3’ ¡UTR ¡ www.omixon.com support@omixon.com
www.omixon.com support@omixon.com
Omixon HLA Twin v1.1.2 www.omixon.com support@omixon.com
Omixon HLA Twin v1.1.2 www.omixon.com support@omixon.com
Quality Control Implemented in HLA Twin www.omixon.com support@omixon.com
Read Quality www.omixon.com support@omixon.com
Coverage www.omixon.com support@omixon.com
Allele Imbalance www.omixon.com support@omixon.com
Fragment Size www.omixon.com support@omixon.com
Noise Ratios www.omixon.com support@omixon.com
Ambiguities 16 14 12 10 3-rd field allele assignment 8 ambiguity DRB1*12:01:01/ 6 *12:10 4 2 0 HLA-A HLA-B HLA-C HLA-DRB1 HLA-DQB1 www.omixon.com support@omixon.com
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