May, 2016 Agenda Introduction Overview of NGS Accreditation with - - PowerPoint PPT Presentation

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May, 2016 Agenda Introduction Overview of NGS Accreditation with - - PowerPoint PPT Presentation

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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.

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NGS Accreditation with EFI Standards, Plus Omixon Update

May, 2016

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
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Agenda

§ Introduction § Overview of NGS Accreditation with EFI Standards § Guest Speakers § Omixon Update

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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)

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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

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NGS workflow

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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

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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

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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

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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

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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

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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

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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

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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

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Guest Speakers

§ Dr. Milena Ivanova § Dr. Brendan Clarke § Dr. Mette Christiansen

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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

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The Evolving NGS Technology 2004-2015

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NGS Platforms

Roche 454 Illumina IonTorrent SOLiD – 5500xl PacBio RS Amplification emPCR bridge PCR emPCR emPCR Non SMS NGS chemistry PS RTs

Semiconductor

Cleavable probe SBL Real Time 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

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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)

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Genetic test in Dept. of Clinical Immunology, University Hospital Alexandrovska

Chimerism monitoring

HLA Typing

Other genetic markers: KIR, MBL, Cytokines, mtDNA, MPN mutations, PID

Solid organ transplantation Diseases Cord blood bank Bone Marrow Donor Registry HSCT

EFI Accredited since 1997

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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

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Work Flow

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

Holotype HLA ™

Hands on time 4h; Total time – 53h 30

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LR-PCR Amplification Strategy

5’ ¡UTR ¡ 6 ¡ 5 ¡ 4 3 ¡ 2 ¡ 1 ¡ 3’ ¡UTR ¡

HLA-­‑DQB1 ¡

5’ ¡UTR ¡ 6 5 4 3 ¡ 2 ¡ 1 ¡ 3’ ¡UTR ¡

HLA-­‑DRB1 ¡

5’ ¡UTR ¡ 6 ¡ 5 ¡ 4 ¡ 3 ¡ 2 ¡ 1 ¡ 7 ¡ 3’ ¡UTR ¡

HLA-­‑A, ¡B, ¡and ¡C ¡(Example ¡is ¡HLA-­‑B) ¡

Full ¡gene ¡characteriza/on ¡ Key ¡region ¡characteriza/on ¡

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Omixon HLA Twin v1.1.2

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Omixon HLA Twin v1.1.2

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Quality Control Implemented in HLA Twin

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Read Quality

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Coverage

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Allele Imbalance

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Fragment Size

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Noise Ratios

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2 4 6 8 10 12 14 16 HLA-A HLA-B HLA-C HLA-DRB1 HLA-DQB1 3-rd field allele assignment ambiguity

DRB1*12:01:01/ *12:10

Ambiguities

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Detection on HLA-C*04 New Allele?

PCR-SBT Patient: C*04, *07 Mother: C*12:03, *07:02

Father: C*04?, *03:04

C*04:01, *07:02 – 1 MM Holotype HLA (Omixon) C*04(new), *07:02:01

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Summary

§ HLA typing by NGS has higher resolution and throughput compared

to Sanger sequencing

§ Commercially available kits provide workflow and software that is

easy to set up.

§ NGS is a good alternative also for laboratories with a lower

throughput. QUALITY IN NGS HLA TYPING

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HLA Typing by NGS – A Change in

Laboratory Praxis

Mette Christiansen, PhD, MSC Department of Clinical Immunology, Aarhus University Hospital, Denmark

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2014 AIM – to Simplify the Workflow

Low resolution

  • 1. Serology (HLA-A & B)
  • 1. PCR-SSO (HLA-A, B, & C)
  • 1. Conventional PCR-SSP
  • 2. PCR-SSP/ SSO

High resolution

  • 1. SBT
  • 1. Reflex-testing to resolve ambiguities
  • 1. PCR-SSP/ Additional SBT primers
  • 2. PCR-SSP/ SSO
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How to Make Radical Changes

Validation Training of staff

  • Change of mindset?

Accreditation Training of staff Routine

  • Change of mindset?
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Results from Validation

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Results from Validation

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Results from Validation

§

10 runs with a total of 240 samples and 1276 loci were analyzed

§

The 2nd field accuracy in passed runs was 99.8%

§

One run did not pass quality control due to over- fragmentation and overload of the MiSeq. – The 2nd field accuracy was 98.8%.

§

All discrepant typings –except

  • ne - were either marked with

red flags in HLA TWIN SW or in the amplification.

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Conclusions

§ Shorter fragments increase the degree of cross-mapping, and reduce

the accuracy.

§ Incomplete amplification result in too many homozygous calls. § Important quality control factors that need to be assessed carefully

are, efficient amplification

– fragment length – cluster density – exon coverage

§ In regard to accreditation these and other parameters are well

described

§ The procedure is robust in regard to reproducibility, accuracy and

contamination.

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Accreditation – Meeting EFI Standards v. 6.3

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2016 GOAL Achieved

Low resolution

  • 1. PCR-SSP HRM
  • 2. NGS/ PCR-SSP

High resolution

  • 1. NGS
  • 2. PCR-SSP
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How to Make Radical Changes

😠Do we need to change anything, it looks

difficult?

😓We don’t have the

time!

😑OK, we will try… 😐We are getting used to it

  • We only have few samples, do we need

to run SBT?

  • We have spare time for new projects.

😑Pooling of samples may delay some typings

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Omixon X2-24/7: The Experience of an Early Adopter Site Brendan Clark Transplant Immunology St James’s University Hospital, Leeds, UK

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2014 HSCT Programme Activity

  • 54 alloHSCT (17 HLA id sib/2 haplo id sib/30 MUD/5 CB)
  • 583 low-res HLA CI types (referral and verification)
  • 460 low-res HLA CII types (referral and verification)
  • 147 hi-res HLA CI types
  • 275 hi-res HLA CII types
  • In-house HLA-A;B;C;DR;DQ PCR-SSP
  • LABtype HLA-A;B;C;DR;DQ PCR-SSO
  • LABtype HD HLA-DRB1
  • Allset Gold HLA-A;B;C;DQB1;DPB1
  • SBT (sendaway)
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HLA Typing HSCT Workflow – The Way We Were

,

Patient HLA- A,B,C,DR,DQ typed by in-house, low-res PCR-SSP, HLA- DR,DQ,DP typed by hi-res PCR-SSP/SSOP Available family members HLA-A,B typed by in-house, low-res PCR-SSP HLA-A,B matched family members HLA-C, DR, DQ typed by in-house, low-res PCR-SSP HLA-matched family members HLA- DR,DQ,DP typed by hi-res PCR-SSP/ SSOP As required patient and HLA matched family members HLA-A,B,C typed by hi-res PCR- SSP to establish intrafamilial HLA identity. Patient and any available HLA matched family member confirmatory typing by LABType low-res PCR-SSOP on fresh samples If no HLA matched family member available perform VUD/cord search. Patient HLA-A,B,C typed by hi-res PCR- SSP. Selected VUDs HLA –A,B,C,DR,DQ,DP typed by hi-res PCR- SSP/SSOP Select family donor Select VUD donor

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Pros Cons

On demand testing Not efficient – iterative testing, up to four rounds of typing required to enable selection of family donor Contains costs Not easily scalable – expansion

  • f clinical programme would

create workload management difficulties Long ‘at-bench’ time for delivery

  • f results

Utilises up to 5 methodological approaches New user specification for typing – HLA-A;B,C,DR,DQ,DP to second field .

Existing Protocol - Evaluation

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Specification for Revised Approach

Omixon Holotype x2 24/7

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‘When was the last time that you actually did any laboratory work?’

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Validation – Phase 1

§ 7 runs involving 168 previously (whole or partially)

second field typed reference DNA’s (EQA, HSCT, 3rd party)

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Validation Phase 1: Investigation of Out-of-Consensus Results

exon incorrect reference type sample id unattributed

n = 53

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Validation – Phase 1 (Revised)

§ 7 runs involving 168 previously (whole or partially)

second field typed reference DNA’s (EQA, HSCT, 3rd party)

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§ 5 parallel runs involving 120 sample DNA’s

Validation - Phase 2

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Validation Phase 2: Investigation of Out-of-Consensus Results

exon sample id unattributed

n=14

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Validation - Phase 2 (Revised)

§ 5 parallel runs involving 120 sample DNS’s

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Ambiguity

A B C DR DQ DP

§ Partial characterisation of DP alleles in IMGT

database § Phasing ambiguity for some combinations

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Holotype - Evaluation

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HSCT Workflow – How We (Almost) Are

Patient and available family members HLA- A,B,C,DR,DQ,DP typed by NGS Patient and any available family member confirmatory HLA- A,B,C,DR,DQ,DP typing by PCR- SSOP/SSP Select family donor If no HLA matched family member available perform VUD/cord search. Selected VUDS HLA- A,B,C,DR,DQ,DP typed by NGS Patient and any available family member confirmatory HLA- A,B,C,DR,DQ,DP typing by NGS

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Next Steps

§ EFI Accreditation § Service Delivery

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Neil Marsden Pamela Hughes Christopher Watson Laura Crinnion Efi Melista Marcello Scala Tim Hague

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CE Mark – Holotype HLA

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CE Marked In Vitro Diagnostic

“The Holotype HLA product is intended for in vitro diagnostic use by professional health care personnel, such as laboratory technicians and physicians, trained in HLA-typing in diagnostic laboratories either EFI or ASHI accredited or able to work according to EFI or ASHI specifications.”

§ Includes full workflow:

– Amplification – Library Prep – Software Interpretation

§ Holotype HLA 24/7 and 96/7 products

– 150bp or 250bp paired end protocol

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Regulatory and Production Practices

CE marked product for In Vitro Diagnostics (IVD)

§

For Europe

Research Use Only (RUO) product

§

For US

§

For registry typing and research All Omixon activities covered by an ISO 13485:2003 Quality Management System (QMS) Manufacturing performed under GMP and ISO 13485

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Holotype HLA -Highlights

§ Flexibility

– 5, 7, 11 locus kits, either 24 or 96 samples per kit

§ Kits can be split up to 3 times, or be combined = 8 to 192 samples per MiSeq run

– Alternating adaptor plates to meet EFI requirements – MiSeq (150 or 250bp data), MiniSeq, NextSeq – Any source of DNA – whole blood, buccal swabs, saliva

§ Scalability

– Two 96 sample adaptor plates = 192 samples per MiSeq run (with 250bp protocol) – Up to 576 samples per run on a NextSeq (coming soon!)

§ Reproducibility

– Early Access Program = 24 labs, 2500 samples – Product Evaluation Study for CE Mark = 200 samples

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Holotype HLA - Highlights

§ Suitability

– Designed by a clinical lab, to be used by clinical labs – CE IVD product – 2500+ samples in clinical routine at CHOP

§ Usability

– Simplest protocol, lowest hands-on time – Automation – Beckman Coulter, Hamilton, Perkin Elmer, TBG, Primadiag

§ Quality

– ISO certified manufacturing, independent manufacturing Quality Control – Most accurate platform (Illumina), superior phasing (paired reads) – 18 Quality Control measures in the software – Highest genotyping accuracy

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Holotype HLA - Early Access Program

§ NGS is new technology - for the HLA clinic § Demonstrating that the technology works reproducibly was a key

component Omixon’s strategy – ‘believe the data’

§ Early Access Program ran from 11/2014 to 6/2015 § 24 labs, 13 countries, 2400 samples, 26000 alleles § >99.7% accuracy and reproducibility

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Holotype HLA - Believe the Data

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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Holotype HLA - Customers

§ More than 75 evaluators § More than 15 labs in clinical production in Europe and the US

– 1 EFI accredited lab – 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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Coming Soon

§ European holding facility

– Reduced shipping times and costs in Europe

§ Holotype HLA - updates

– 576 samples per run on the NextSeq – CE mark applied to 11 locus kits (Q1 2017)

§ Monotype HLA

– New Product – HLA-B and DQB/A only

§ HLA Explore

– New Product – RUO software, for big data and long read data

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Omixon –You Tube Channel

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Omixon Academy Microsite

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Marcello Scala Director of Sales, EMEA

marcello.scala@omixon.com

Tim Hague Chief Executive Officer

tim.hague@omixon.com