Introduction to GENDX HLA typing products

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

GENDX Company tests and services, analysis software and education 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 Single PCR Coverage of essential regions # Class I; entire gene amplified # Class II; exon 2 & 3 and beyond Unsurpassed ambiguity resolving power # 2nd 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 CE-IVD 6

SBTengine® Software, CE-IVD Powerful HLA-typing software using SBT data 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 CE-IVD 7

Sequencing Based Typing 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 CE-IVD 8

SBT Strategies Core typing: sequencing essential regions HLA-A, B: Exons 2,3 and 4 HLA-C: Exons 2 and 3 DRB: Exon 2, DRB4 also exon 3 DQB1: Exons 2 and 3 DPB1: Exon 2 9 9

HLA Nomenclature HLA-A*02:01:01:01 HLA-A2 Serology HLA-A*02 Allele group main specificity/ serological equivalent HLA-A*02:01 Allelic subtypes: differences in 3rd and 4th digit = different protein HLA-A*02:01:01 Non-coding variation Differences in 5th and 6th digit = different nucleotides, identical protein Silent variation HLA-A*02:01:01:01 Variation outside coding regions Differences in 7th and 8th digit = variation outside coding regions Intron variation, splice sites, promotor 10 10

HLA Nomenclature Additional characters: N Null alleles HLA-A*24:09N No expression Premature stop codon Splice sites L Low expression: HLA-A*24:02:01:02L S Secreted: HLA-B*44:02:01:02S Q Questionable expression: HLA-A*32:11Q Unconfirmed aberrant expression 11 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 13

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

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

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

Example HLA-B, Core + GSSP’s SBTexcellerator® GSSP B20 + B26 + B16 All ambiguities resolved ! CE-IVD 17 17

HLA-A Strategy CE-IVD

HLA-B Strategy CE-IVD

HLA-C Strategy CE-IVD

HLA-DRB1 Strategy CE-IVD

HLA-DQB1 Strategy CE-IVD

HLA-DRB3/4/5 Strategy CE-IVD

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

SBTexcellerator® Single PCR reaction per HLA locus For HLA Class I coverage of the complete gene For HLA Class II coverage of exon 2 (and exon3) Primers provided for amplification and sequencing Core regions: Extended regions to resolve allele ambiguities GSSP’s to resolve genotype ambiguities CE-IVD 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 A reagent box contains amplification primers for: HLA loci A, B, C, DRB1 and DQB1 Single PCR reaction per HLA gene Sufficient reagent for amplication of 24 samples for all 5 loci Amplification is performed with Qiagen LongRange 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 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 MiSeq One button analysis For research use only 29

Custom laboratory service HLA-typing service for research and diagnostics Research projects New alleles # Validation & submission Registry typing Confirmatory typing Diagnostics ISO 13485 certified ISO 13485 certified 30

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