Introduction to GENDX HLA typing products 1

Introduction to GENDX HLA typing products 1

The GENDX Mission Gen. Dx develops and markets innovative, CE marked diagnostic products, providing crucial information for patient-tailored treatments in and beyond transplantation therapy. 2

GENDX Company • Develops and markets a comprehensive line of IVD tests and services, analysis software and education • • Located in Utrecht, Netherlands Spin-off from University Medical Centre Utrecht • Founded in 2005 • ISO 13485: 2003 quality system • CE-IVD marked products 3

GENDX Products • SBTexcellerator® Reagents, CE-IVD Accelerating HLA typing by excellent performance • SBTengine® Software, CE-IVD Powerful HLA-typing software using SBT data • NGS-go® Reagents, RUO Amplification strategy for down stream NGS applications • NGSengine® Software, RUO The Ultimate software tool for NGS HLA-typing 4

GENDX Products • Custom laboratory service HLA-typing service for research and diagnostics • Education HLA-SBT Training School, ABHI accreditation • Production Facility For small-medium scale production of DNA diagnostics ISO 13485 certified 5

SBTexcellerator® Reagents, CE-IVD Accelerating High Resolution HLA Typing by Excellent Performance 1. 2. 3. 4 5 6 CE-IVD Single PCR Coverage of essential regions # Class I; entire gene amplified # Class II; exon 2 & 3 and beyond Unsurpassed ambiguity resolving power # 2 nd Generation GSSP # DARTTM in combination with SBTengine® Automated handling of null alleles in combination with SBTengine® Easy integration in your laboratory due to flexible kit design Primers; Total Solution approach to type 50 DNA samples 6

SBTengine® Software, CE-IVD Powerful HLA-typing software using SBT data 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. CE-IVD Highly automated, fast and accurate allele assignment Easy-of-use through uncluttered user interface GSSP Predicting System (GPS) for selecting GSSP’s upfront – pre typing info Filtering options of crucial positions- saves analysis time XML-based reports Convenient network licensing available for multi-user sites New allele reporting Reagent independent Automatic updates of software and libraries Fast, knowledgeable customer support 7

Sequencing Based Typing • • • CE-IVD High-resolution method Uses all nucleotides to determine HLA alleles No changes in strategy needed with IMGT/HLA update Detection of new alleles Limited number of reactions 8

SBT Strategies • Core typing: sequencing essential regions • Essential regions: – – – HLA-A, B: HLA-C: DRB: DQB 1: DPB 1: Exons 2, 3 and 4 Exons 2 and 3 Exon 2, DRB 4 also exon 3 Exons 2 and 3 Exon 2 9

HLA Nomenclature HLA-A*02: 01: 01 • HLA-A 2 Serology • HLA-A*02 Allele group – main specificity/ serological equivalent – differences in 3 rd and 4 th digit = different protein – – Differences in 5 th and 6 th digit = different nucleotides, identical protein Silent variation – – Differences in 7 th and 8 th digit = variation outside coding regions Intron variation, splice sites, promotor • HLA-A*02: 01 Allelic subtypes: • HLA-A*02: 01 Non-coding variation • HLA-A*02: 01: 01 Variation outside coding regions 10

HLA Nomenclature Additional characters: • N – – – • L • S • Q – Null alleles HLA-A*24: 09 N No expression Premature stop codon Splice sites Low expression: HLA-A*24: 02: 01: 02 L Secreted: HLA-B*44: 02: 01: 02 S Questionable expression: HLA-A*32: 11 Q Unconfirmed aberrant expression 11

SBT Strategy SBTexcellerator® Class I Amplification primer with QIAGEN Long Range Polymerase Core Sequencing primer Extended Region Sequencing primer Group Specific Sequencing Primer CE-IVD 12

Example HLA-C SBTexcellerator® Amplification primer with QIAGEN Long Range Polymerase Core Sequencing primer CE-IVD 13

HLA-C, Core SBTexcellerator® - SBTengine® CE-IVD Allele ambiguities 14

HLA-C, Core + Extended SBTexcellerator® - SBTengine® All ambiguities resolved ! CE-IVD 15

Example HLA-B, Core SBTexcellerator® - SBTengine® CE-IVD Genotype ambiguity 16

Example HLA-B, Core + GSSP’s SBTexcellerator® GSSP B 20 + B 26 + B 16 All ambiguities resolved ! CE-IVD 17

HLA-A Strategy CE-IVD 18

HLA-B Strategy CE-IVD 19

HLA-C Strategy CE-IVD 20

HLA-DRB 1 Strategy CE-IVD 21

HLA-DQB 1 Strategy CE-IVD 22

HLA-DRB 3/4/5 Strategy CE-IVD 23

Reagent Box Set-up SBTexcellerator® 5’UTR 1 CORE CE-IVD 2 3 4 EXTENDED 5 6 7 8 3’UTR FULL FU 24

SBTexcellerator® • • CE-IVD Single PCR reaction per HLA locus For HLA Class I coverage of the complete gene For HLA Class II coverage of exon 2 (and exon 3) Primers provided for amplification and sequencing – Core regions: – Extended regions to resolve allele ambiguities – GSSP’s to resolve genotype ambiguities 25

Next Generation Sequencing Typing Features • One pass method • Limited ambiguities – No need for GSSP’s and or specific sequencing primers • Bulk method – pooling of samples For research use only 26

NGS-go® Reagents Amplification strategy for down stream NGS applications 1. A reagent box contains amplification primers for: 2. 3. 4. 5. Single PCR reaction per HLA gene Sufficient reagent for amplication of 24 samples for all 5 loci Amplification is performed with Qiagen Long. Range PCR kit Coverage of essential regions # Class I ; entire gene amplified # Class II; complete coverage of relevant coding regions, extending beyond exon 2 & 3 where necessary Complete exon readability Amplicon can be used for amplicon sequencing with various available NGS platforms 6. 7. HLA loci A, B, C, DRB 1 and DQB 1 For research use only 27

NGSengine® Software The Ultimate software tool for NGS HLA-typing Saves Time • High percentage of analysed reads • Automated HLA locus identification • Typing of heterozygous samples in one pass • Crucial position toggling • Easy handling of missing data Improves results • Clear quality statistics • Genotype references • Whole gene representation • Phasing visualisation For research use only 28

NGSengine® Software The Ultimate software tool for NGS HLA-typing Easy implementation • NGS platform independent! Optimized for data from Roche 454, Ion Torrent and Mi. Seq • One button analysis For research use only 29

Custom laboratory service HLA-typing service for research and diagnostics 1. 2. 3. 4. 5. 6. Research projects New alleles # Validation & submission Registry typing Confirmatory typing Diagnostics ISO 13485 certified 30

Education HLA-SBT Training School, ABHI accreditation 1. HLA-SBT Training Sessions - 1 day Maastricht, The Netherlands, Saturday 11 th May 2012. Just prior to EFI 2. HLA-SBT Training School - 2½ day - Including wet laboratory practice Utrecht, The Netherlands March 2013 Register at: www. Gen. Dx. com ISO 13485 certified 31

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