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Erik H. Rozemuller - One of the best experts on this subject based on the ideXlab platform.

  • P101 Chimerism monitoring by next-generation sequencing: A multiplatform comparison
    Human Immunology, 2018
    Co-Authors: Evelien E. Bouwmans, Maarten T. Penning, Loes A. Van De Pasch, Erik H. Rozemuller
    Abstract:

    Aim Accurate monitoring of the chimeric status after stem cell transplantation is essential for early detection of relapse and at the moment mainly performed by STR or qPCR. The major disadvantages of STR are the laborious data analysis and poor sensitivity. The qPCR technique enables a much quicker workflow and better sensitivity. However a disadvantage is the need of a pre-transplant sample for each monitoring event. Performing chimerism monitoring by NGS will eliminate these limitations while allowing for a multiplexed setup, reducing the amount of needed lab work and DNA. The workflow can be combined with routinely applied NGS HLA typing in clinical laboratories. Methods PCR amplification primers have been designed for a set of biallelic markers. These markers were tested on a number of artificial chimeric samples in a range between 0.01–100% of positive DNA mixed with negative DNA. Amplicons were applied in the respective NGSgo library preparation workflows and run on Illumina MiSeq, Ion Torrent S5 and Oxford Nanopore MinION systems. NGS data was analysed with customized analysis tools designed to quantitate the two variants of each marker. Results Each marker was tested on a small panel of non-chimeric samples (Coriell Institute) and resulted in clear homo- or heterozygous patterns for all markers. Results of the different platforms on the artificial chimeric samples in a range of 0.01%–100% were compared. Sensitivity was determined for each sequencing platform. A read depth threshold was established for each of the platforms to achieve a minimum sensitivity of 0.05%. Results including difference between sequencing platforms will be discussed further. Conclusions The results demonstrate that NGS-based chimerism monitoring is feasible for a wide range of NGS platforms, with a high sensitivity and wide dynamic range. E.E. Bouwmans: 5. Employee; Company/Organization; GenDx. L.A. van de Pasch: 5. Employee; Company/Organization; GenDx. M.T. Penning: 5. Employee; Company/Organization; GenDx. E. Rozemuller: 5. Employee; Company/Organization; GenDx. 6. Stock Shareholder; Company/Organization; GenDx. 7. Other (Identify); Company/Organization; Founder of GenDx.

  • OR36 NGS Allele-level typing strategy for nine KIR genes
    Human Immunology, 2018
    Co-Authors: Loes A. Van De Pasch, Maarten T. Penning, Karin Van Ham, Sanne Vendelbosch, Erik H. Rozemuller
    Abstract:

    Aim Killer-cell Immunoglobulin-like Receptors (KIRs) are encoded by 17 genes in the Leukocyte Receptor Complex (LRC). These genes are highly polymorphic, on the level of structural variants, and copy-number variation. These characteristics make it challenging to design specific amplifications, and to perform reliable analysis of each KIR allele present in a sample. To enable allele-level resolution KIR genotyping, we developed an NGS-based strategy that involves whole gene amplification of KIR genes. Methods We developed KIR-gene specific amplification primers, with specificity for one or more KIR genes (KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR3DS1, KIR3DL1, KIR3DL2, KIR3DL3 and KIR3DP1). Amplicons were processed in the NGSgo® (GenDx) library preparation procedure (GenDx) and paired-end sequenced on an Illumina MiSeq platform (2 × 151 bp). Data was genotyped with NGSengine® software (GenDx), using KIR database IPD-KIR 2.7.1. Results Multiple samples with known and unknown KIR genotypes were analyzed using this new strategy. KIR amplification, sequencing and subsequent typing was successful for the majority of samples. Many samples could be typed unambiguously, despite the fact that phasing was not fully accomplished due to the length of the genes (up to 17 kb). Remaining challenges in analysis are samples with KIR copy number variation and high degree of homology, which can hamper the data analysis. Conclusions KIR genotyping at allelic level is feasible using the short read sequencing technology of Illumina. This new typing strategy may be an attractive alternative to existing KIR genotype assays that only determine KIR gene content at a limited resolution, or complex sequencing methods aiming to sequence the entire LCR. L.A. van de Pasch: 5. Employee; Company/Organization; GenDx. K. van Ham: 5. Employee; Company/Organization; GenDx. S. Vendelbosch: 5. Employee; Company/Organization; GenDx. M.T. Penning: 5. Employee; Company/Organization; GenDx. E.H. Rozemuller: 5. Employee; Company/Organization; GenDx. 6. Stock Shareholder; Company/Organization; GenDx. 7. Other (Identify); Company/Organization; Founder of GenDx.

  • P080 HLA disease association assignment by multiplexed NGS assay
    Human Immunology, 2016
    Co-Authors: Nienke Westerink, Erik H. Rozemuller, Inge Van Rooy, Claudia Rebel, Michelle Van Heck, Maarten T. Penning
    Abstract:

    Aim Strong association of HLA molecules with autoimmune and inflammatory diseases have been identified, strengthening the value of HLA genetic screening for diagnostic purposes. There’s a strong association between Ankylosing Spondylitis (AS) and HLA-B27. In ∼90% of European patients, B27 alleles strongly predispose for AS. In ∼90% of patients with Coeliac Disease, the HLA-DQ2.5 phenotype is expressed, encoding HLA-DQA1∗05:01 and DQB1∗02:01 alleles, with the remaining 10% mostly expressing the HLA-DQ8 molecule, encoding the DQA1∗03 variant and DQB1∗03:02 alleles. Also in the field of pharmacogenetics there’s a growing interest in the role of HLA. HLA-B∗57:01 screening to prevent Abacavir hypersensitivity syndrome is now a routine clinical use in the developed world. We developed a novel NGS-based genetic screening test that enables 2nd field resolution typings of HLA-B, DQA1, and DQB1 in a single tube. Methods A multiplexed amplification assay was established that encompass HLA-B, HLA-DQA1 and HLA-DQB1 locus-specific amplification of exon 2 and 3 in a single tube. The assay was compatible with the multiplex PCR kit (Qiagen) and required a PCR enhancer (GenDx). Analytical performance studies were assessed including screening of a large gDNA reference panel (n = 96 samples) to verify robustness. Amplicons were pooled and processed into the NGSgo workflow for Illumina (GenDx) and analyzed by NGSengine software (GenDx). Results For all samples tested (n = 96) strong amplicons of the expected sizes were generated in a multiplexed PCR. NGS data showed high locus mappability (>94%), and full coverage of the amplicon with even distribution of reads, especially for HLA-B, and HLA-DQA1, having average read depths >1500. For all samples, HLA-B, HLA-DQB1 and HLA-DQA1 typings were obtained that are in concordance with the pre-types. The multiplexed assay showed to be robust and capable of detecting both alleles when present in a balanced manner. Conclusions We developed a novel NGS-based genetic screening test that enables HLA-B, DQA1, and DQB1 typings in a single tube. The genetic screening test proved to be robust and useful for pharmacogenetic screening of HLA-B alleles and identification of autoimmune disease associated genetic susceptibilities like celiac disease. N. Westerink:  Employee; Company/Organization; GenDx.I. van Rooy:  Employee; Company/Organization; Gendx. M. Van Heck:  Employee; Company/Organization; GenDx. C. Rebel:  Employee; Company/Organization; GenDx. E. Rozemuller:  Stock Shareholder; Company/Organization; GenDx. M. Penning:  Employee; Company/Organization; GenDx.

  • High-throughput and cost-effective NGS strategy for intermediate-resolution HLA typing
    Human Immunology, 2015
    Co-Authors: Inge Van Rooy, Michelle Bacelar, Erik H. Rozemuller, R Kooter, Claudia Rebel, Roel Van Aard, Bram Luiken, Loes A. Van De Pasch, Evelien E. Bouwmans, Maarten T. Penning
    Abstract:

    Aim The National Marrow Donor Program (NMDP) requires that each newly recruited donor is typed at intermediate resolution level for HLA-A, -B, -C, -DRB1 and optionally -DQB1. Therefore we developed an NGS assay specifically designed for intermediate resolution HLA typing (class I exon 2 & 3, and class II exon 2) that is suitable for high-throughput. PCR conditions were optimized such to reduce the costs of reagents. Moreover, the limited size of the amplicon allows for an increased sample capacity per run on either the MiSeq or Ion Torrent platform: a four-fold increase on average compared to the whole-gene amplification strategy. Method We integrated this NGSessenz strategy into the previously developed NGSgo workflows for MiSeq and Ion Torrent platforms. For the MiSeq platform, high-throughput and reproducibility was verified by running 4 × 96 DNA samples on a micro flow cell. For IonTorrent, we verified the capacity of the smallest (314 chip) and the largest (318 chip) by running a variety of samples. For each sample, loci were pooled prior to the execution of the NGSgo platform-specific library preparation and subsequent NGS procedures were performed. Results We will present the NGS data that were obtained for the robustness of amplification, mappability of the reads, read depth per locus, concordance of NGS typing with reference type, reproducibility of the NGS data generated, and the capacity of the NGSessenz assay on the different platforms. Conclusions Taken together, we will demonstrate the major advantage of this new NGSessenz strategy in ease-of-use and cost-effectiveness for HLA registry typing by means of NGS. I. Van Rooy: Employee; Company/Organization; GenDx. C. Rebel: Employee; Company/Organization; GenDx. R. Van Aard: Employee; Company/Organization; GenDx. M. Bacelar: Employee; Company/Organization; GenDx. B. Luiken: Employee; Company/Organization; GenDx. L. Van de Pasch: Employee; Company/Organization; GenDx. E. Bouwmans: Employee; Company/Organization; GenDx. R. Kooter: Employee; Company/Organization; GenDx. E. Rozemuller: Stock Shareholder; Company/Organization; GenDx. M. Penning: Employee; Company/Organization; GenDx. W. Mulder: Stock Shareholder; Company/Organization; GenDx. N. Westerink: Employee; Company/Organization; GenDx.

  • High-resolution HLA typings obtained for high-throughput NGS using the Illumina MiSeq platform
    Human Immunology, 2015
    Co-Authors: Loes A. Van De Pasch, Michelle Bacelar, Maarten T. Penning, Erik H. Rozemuller, R Kooter, Claudia Rebel, Roel Van Aard, Evelien E. Bouwmans, Nienke Westerink
    Abstract:

    Aim Next-generation sequencing (NGS) technology generates sequences from single DNA molecules that enables unique identification of the paternal and maternal alleles. The NGS technology has great potential in the application of HLA typing for diagnostic purposes and generating reliable, unambiguous HLA typing results in a high-throughput fashion. Here we present data obtained for 11 HLA loci using a HLA typing assay dedicated for high-throughput NGS-based HLA typing on the Illumina MiSeq. The implementation of this high-throughput NGS strategy would be highly advantageous to obtain robust high-resolution HLA typing results for registry labs and routine diagnostic purposes. Method A large genomic DNA reference panel, including UCLA reference samples, were selected for high-throughput NGS HLA typing. The whole gene of HLA-A, -B, -C, and the essential genomic regions of HLA-DRB1, -DRB345, -DQA1, -DQB1, -DPA1 and -DPB1 were PCR-amplified in a single PCR for each locus. Amplicons of each sample were pooled and processed using our NGSgo® DNA library preparation workflow for Illumina MiSeq. During library preparation the samples were fragmented, barcoded and multiplexed. Finally paired-end sequencing (2x150 cycles) was performed on the Illumina MiSeq platform using a standard flow cell and data was analysed using our HLA typing software NGSengine®. Results Paired-end NGS data was obtained for 11 HLA loci (class I and II) of most genomic DNA reference samples in a single Illumina MiSeq sequencing run. The NGS data covered the complete HLA region of interest by highly accurate measures for all HLA loci tested. All HLA alleles were effectively separated and, in most cases, could be phased throughout the gene. For the majority of samples, unambiguous third- and fourth-field resolution typing were obtained for all loci that are in concordance with the second-field or higher reference types. Conclusions The high-throughput NGSgo® strategy that we have developed demonstrates to be a powerful method to perform high-resolution HLA typing by means of NGS in a robust and reliable manner. L. Van de Pasch: Employee; Company/Organization; GenDx. M. Bacelar: Employee; Company/Organization; GenDx. C. Rebel: Employee; Company/Organization; GenDx. R. Van Aard: Employee; Company/Organization; GenDx. R. Kooter: Employee; Company/Organization; GenDx. E. Rozemuller: Stock Shareholder; Company/Organization; GenDx. M. Penning: Employee; Company/Organization; GenDx. E. Bouwmans: Employee; Company/Organization; GenDx. N. Westerink: Employee; Company/Organization; GenDx.

Medhat Askar - One of the best experts on this subject based on the ideXlab platform.

  • p099 comparison of ssop versus ngs for typing of hla a b c drb1 drb3 b4 b5 dqa1 dqb1 dpa1 dpb1 toward single pass high resolution hla typing in support of solid organ and hematopoietic cell transplant programs
    Human Immunology, 2018
    Co-Authors: Anajane G. Smith, Shalini Pereira, Wyatt Nelson, Andres Jaramillo, Faisal Khan, Noureddine Berka, Marcelo J Pando, Maria Bettinotti, Medhat Askar
    Abstract:

    Aim Many laboratories use a 2-tier HLA typing process: a first pass intermediate resolution and then high resolution, when clinically necessary. SSP or qPCR provide rapid typing for deceased donor workup; SSOP is widely employed for higher volumes; and Sanger sequencing has been the gold standard for high resolution. However, SSOP and Sanger SBT often yield ambiguous results and resolution is expensive and time consuming. Next generation sequencing offers a single pass technology to achieve unambiguous HLA allele assignments. We compared commercial SSOP and NGS systems with respect to accuracy, turnaround time, effort and resolution level. Methods Five laboratories submitted coded, blinded samples, previously typed by SSOP at each institute, to a separate lab for NGS of HLA-A, B, C at exons 1–7, DRB1, DRB3/4/5, DQA1, DQB1, DPB1 at exons 1–4, and DPA1 at exons 2–4. For up to 24 samples by SSOP or up to 48 samples by NGS, benchwork is completed by one technologist on day 1 with results available for reporting on day 2. Results At the SSOP resolution level, results were concordant except for 11 SSOP assignments in 8 specimens due to false probe reactions at DRB1, DRB5, DQA1, DQB1, and DPB1. NGS identified 21 novel sequences: one with a multi-exon deletion and 20 with SNP polymorphisms in HLA-C, DPA1, DPB1, DQA1, DRB1, DRB3, DRB4, and DRB5 alleles. Across the highly polymorphic HLA-A, B, C, DRB1 loci, only 1% of intermediate resolution, IR, SSOP results were unambiguous. Even higher resolution, HR, SSOP by 1 lab gave only 17% specific allele assignments. In contrast, over 99% of NGS results were specific unambiguous genotypes. Across all loci, NGS typing was specific except for certain discrete diploid ambiguities, also found in SSOP. Conclusions This study provides a compelling rationale for implementing NGS for single pass HLA analysis with accurate, 2 day turnaround, which includes the high resolution, unambiguous genotyping critical for unrelated donor HCT and that may be required for sensitized patients with allele specific antibodies. A.G. Smith: 2. Consultant; Company/Organization; Scisco Genetics Inc. S.E. Pereira: 4. Scientific/Medical Advisor; Company/Organization; Scisco Genetics Inc. C. Pyo: 4. Scientific/Medical Advisor; Company/Organization; Scisco Genetics Inc. W. Nelson: 5. Employee; Company/Organization; Scisco Genetics Inc. M. Bettinotti: 3. Speaker’s Bureau; Company/Organization; One Lambda ThermoFisher. M.Z. Askar: 3. Speaker’s Bureau; Company/Organization; Immucor. 4. Scientific/Medical Advisor; Company/Organization; Illumina. D.E. Geraghty: 6. Stock Shareholder; Company/Organization; Scisco Genetics Inc. Download high-res image (283KB) Download full-size image

  • OR27. Novel next generation sequencing based chimerism assay for engraftment monitoring in hematopoietic cell transplantation
    Human Immunology, 2018
    Co-Authors: Medhat Askar, Anajane G. Smith, Shalini Pereira, Chul-woo Pyo, Amanda Willis, Jenifer D. Williams, Leah Pittmon, Wyatt C. Nelson, Daniel E. Geraghty
    Abstract:

    Aim Most laboratories providing chimerism testing use electrophoretic analysis of STR loci or qPCR of SNP polymorphisms. STR assays are limited to 1–5% sensitivity and qPCR requires substantial amounts of DNA which may not be feasible in patients with low blood cell counts or for analysis of rare cell subsets. We developed a chimerism assay based on NGS of SNP and InDel polymorphisms and validated it in a clinical transplant immunology lab supporting a large HCT program. Methods Multiple polymorphic loci were evaluated for specificity, sensitivity and accuracy using artificial admixtures as well as direct comparison to qPCR results from patient specimens post HCT. An initial panel comprised 37 SNPs on 18 chromosomes and 12 InDels on 10 chromosomes. Target generation is performed in 2 multiplexed PCRs with 5 ng sample DNA per reaction. All NGS benchwork is accomplished within 6 h, followed by a 7 h MiSeq run, so that results are available for reporting early on day 2. Results Initial NGS testing used artificial admixtures of unrelated individuals, with mixtures from 10% to 0.1%, see Table 1. On average, 30 informative markers were identified between unrelated individuals and NGS achieved 0.5% sensitivity among all 4 admixture sets. Table 2 shows the results for NGS versus qPCR for 7 patient post HCT specimens. Download : Download high-res image (334KB) Download : Download full-size image Download : Download high-res image (247KB) Download : Download full-size image Conclusions These preliminary results show that NGS technology can provide accurate, informative, and sensitive detection of a minority species with minimal DNA quantity requirements, rapid turnaround and minimal hands on time. As laboratories implement NGS technology for high resolution genotyping of HLA and other genetic loci, extension to engraftment monitoring might also offer workplace efficiencies. M.Z. Askar: 3. Speaker’s Bureau; Company/Organization; Immucor. 4. Scientific/Medical Advisor; Company/Organization; Illumina. S.E. Pereira: 4. Scientific/Medical Advisor; Company/Organization; Scisco Genetics Inc. A.G. Smith: 2. Consultant; Company/Organization; Scisco Genetics Inc. C. Pyo: 4. Scientific/Medical Advisor; Company/Organization; Scisco Genetics Inc. W. Nelson: 5. Employee; Company/Organization; Scisco Genetics Inc. D.E. Geraghty: 6. Stock Shareholder; Company/Organization; Scisco Genetics Inc.

  • P099 Comparison of ssop versus ngs for typing of HLA-A, B, C, DRB1, DRB3/B4/B5, DQA1, DQB1, DPA1, DPB1: Toward single pass high resolution hla typing in support of solid organ and hematopoietic cell transplant programs
    Human Immunology, 2018
    Co-Authors: Anajane G. Smith, Shalini Pereira, Andres Jaramillo, Faisal Khan, Noureddine Berka, Marcelo J Pando, Maria Bettinotti, Chul-woo Pyo, Wyatt C. Nelson, Medhat Askar
    Abstract:

    Aim Many laboratories use a 2-tier HLA typing process: a first pass intermediate resolution and then high resolution, when clinically necessary. SSP or qPCR provide rapid typing for deceased donor workup; SSOP is widely employed for higher volumes; and Sanger sequencing has been the gold standard for high resolution. However, SSOP and Sanger SBT often yield ambiguous results and resolution is expensive and time consuming. Next generation sequencing offers a single pass technology to achieve unambiguous HLA allele assignments. We compared commercial SSOP and NGS systems with respect to accuracy, turnaround time, effort and resolution level. Methods Five laboratories submitted coded, blinded samples, previously typed by SSOP at each institute, to a separate lab for NGS of HLA-A, B, C at exons 1–7, DRB1, DRB3/4/5, DQA1, DQB1, DPB1 at exons 1–4, and DPA1 at exons 2–4. For up to 24 samples by SSOP or up to 48 samples by NGS, benchwork is completed by one technologist on day 1 with results available for reporting on day 2. Results At the SSOP resolution level, results were concordant except for 11 SSOP assignments in 8 specimens due to false probe reactions at DRB1, DRB5, DQA1, DQB1, and DPB1. NGS identified 21 novel sequences: one with a multi-exon deletion and 20 with SNP polymorphisms in HLA-C, DPA1, DPB1, DQA1, DRB1, DRB3, DRB4, and DRB5 alleles. Across the highly polymorphic HLA-A, B, C, DRB1 loci, only 1% of intermediate resolution, IR, SSOP results were unambiguous. Even higher resolution, HR, SSOP by 1 lab gave only 17% specific allele assignments. In contrast, over 99% of NGS results were specific unambiguous genotypes. Across all loci, NGS typing was specific except for certain discrete diploid ambiguities, also found in SSOP. Conclusions This study provides a compelling rationale for implementing NGS for single pass HLA analysis with accurate, 2 day turnaround, which includes the high resolution, unambiguous genotyping critical for unrelated donor HCT and that may be required for sensitized patients with allele specific antibodies. A.G. Smith: 2. Consultant; Company/Organization; Scisco Genetics Inc. S.E. Pereira: 4. Scientific/Medical Advisor; Company/Organization; Scisco Genetics Inc. C. Pyo: 4. Scientific/Medical Advisor; Company/Organization; Scisco Genetics Inc. W. Nelson: 5. Employee; Company/Organization; Scisco Genetics Inc. M. Bettinotti: 3. Speaker’s Bureau; Company/Organization; One Lambda ThermoFisher. M.Z. Askar: 3. Speaker’s Bureau; Company/Organization; Immucor. 4. Scientific/Medical Advisor; Company/Organization; Illumina. D.E. Geraghty: 6. Stock Shareholder; Company/Organization; Scisco Genetics Inc. Download : Download high-res image (283KB) Download : Download full-size image

  • OR47 The mystery of the missing DRB1 allele – solved by NGS!!!
    Human Immunology, 2018
    Co-Authors: Anajane G. Smith, Shalini Pereira, Medhat Askar, Chul-woo Pyo, Amanda Willis, Wyatt C. Nelson, Daniel E. Geraghty
    Abstract:

    The mystery began when HLA typing, of two apparently unrelated individuals at two different institutions, identified the presence of a DRB5∗01 gene without any evidence of an expected linked DRB1∗15, DRB1∗16, or, in rare cases, DRB1∗01 allele. Historic data from Sanger sequence-based typing included DRB1∗14, DRB3∗02:02, DRB5∗01:01, DQB1∗05:03, 06:02 for Individual 1 and DRB1∗04:01; DRB4∗01; DRB5∗01:01; DQB1∗03:01, 06:02 for Individual 2. Since those analyses only assessed exon 2, the apparent absence of a DRB1 allele associated with the DRB5 gene raised the possibility of an entire linked DRB1 gene having gone missing. Both specimens were recently re-examined using next generation sequencing technology. HLA-A, B, C genes were assessed for exons 1–7; DRB1, DRB3/B4/B5, DQA1, DQB1, and DPB1 were assessed for exons 1–4; and DPA1 for exons 2–4. In Individual 1, NGS identified DRB1∗14:54:01 and a DRB1∗15 exon 1 sequence, but without any evidence of DRB1∗15 sequences for exons 2 and 3. Since the exon 4 sequences of DRB1∗14:54:01 and DRB1∗15 are identical, it could not be determined whether the DRB1∗15 exon 4 sequence was present. Individual 2, typed as DRB1∗04:01:01 and, again, a DRB1∗15 exon 1. In the latter case, it was clear that no exon 2, 3, or 4 sequences were present for the DRB1∗15 gene, since exon 4 sequences differ between DRB1∗04:01:01 and DRB1∗15. The table shows the full NGS typing data. The HLA alleles shared by both individuals suggest a putative extended haplotype: A∗25:01:01-C∗12:03:01-B∗18:01:01-DRB1∗15new-DRB5∗01:01:01-DQA1∗01:02:01-DQB1∗06:02:01-DPA1∗01:03:01-DPB1∗23:01:01. NGS typing solved the missing allele mystery through identification of a truncated DRB1∗15 allele comprising an exon 1 sequence but without exons 2, 3, or 4. Similarly, there is DRB4∗03:01N which has an exon 3 sequence of a DRB4∗01:01, but without an exon 2 sequence. These unusual alleles appear to be the results of historic multi-exon deletion events and add to the astounding diversity of the human major histocompatibility complex. Download high-res image (242KB) Download full-size image A.G. Smith: 2. Consultant; Company/Organization; Scisco Genetics Inc. S.E. Pereira: 4. Scientific/Medical Advisor; Company/Organization; Scisco Genetics Inc. C. Pyo: 4. Scientific/Medical Advisor; Company/Organization; Scisco Genetics Inc. W. Nelson: 5. Employee; Company/Organization; Scisco Genetics Inc. M.Z. Askar: 3. Speaker’s Bureau; Company/Organization; Immucor. 4. Scientific/Medical Advisor; Company/Organization; Illumina. D.E. Geraghty: 6. Stock Shareholder; Company/Organization; Scisco Genetics Inc.

  • OR04: A MULTI-CENTER STUDY USING NEXT-GENERATION SEQUENCING (NGS) FOR HLA GENOTYPING
    Human Immunology, 2014
    Co-Authors: Curt Lind, Medhat Askar, Deborah Ferriola, Anh Huynh, Jamie L. Duke, Anna Papazoglou, Attila Berces, Mette Christiansen, Wei Dong, Manish J. Gandhi
    Abstract:

    Aim NGS is well-suited for HLA typing as it can deliver highly accurate and unambiguous results. A simplified protocol has been developed for use with the Illumina MiSeq and has been subjected to an inter- Methods: Six laboratories, each with varying levels of NGS experience, participated in this double-blinded study. The same 16 samples were typed at HLA-A, B, C, DRB1, and DQB1 by each lab. The protocol consisted of LR-PCR, library prep, and paired-end 250 bp sequencing. Two indexing strategies were employed: (1) Locus-specific indexing by which each locus was tagged uniquely and (2) sample-specific indexing whereby all 5 loci for a sample were pooled prior to library prep. Sequence analysis was performed with Target HLA (Omixon) and NGSengine (GenDx) to assess complementarity of two analysis algorithms. Results Six sequencing runs yielded an average output of 7.8 Gb per run. The average number of sequence reads per library was 387,813. However, analysis was limited to 40K reads for the locus-indexed libraries and 200K reads for the sample-indexed libraries (amplicon pools) resulting in an average coverage of 1444. A sufficient number of reads for genotype analysis were obtained for 98.4% of libraries. Genotype accuracy is shown in Table 1. Reproducibility between labs was 99.7%. The only cause of discordance between labs was cross-contamination of a single amplicon. Conclusion The protocol developed for the MiSeq is reproducible, highly accurate, and its simplicity makes it amenable to clinical testing. The ability of the software to decipher reads from multiple loci in a single library demonstrates the potential for using an amplicon pooling approach that further simplifies sample prep and reduces expense. As demonstrated in this study the protocol is suitable for moderate test volume. However, since 100 samples can be typed in a single run using this approach. A. Berces: Employee; Company/Organization; Omixon, Inc. T. Hague: Employee; Company/Organization; Omixon, Inc. G. Horvath: Consultant; Company/Organization; Omixon, Inc. R. Kooter: Employee; Company/Organization; GenDx . W. Mulder: Stock Shareholder; Company/Organization; GenDx . M. Penning: Employee; Company/Organization; GenDx. K. Rigo: Employee; Company/Organization; Omixon, Inc. E. Rozemuller: Stock Shareholder; Company/Organization; GenDx.

Maarten T. Penning - One of the best experts on this subject based on the ideXlab platform.

  • OR36 NGS Allele-level typing strategy for nine KIR genes
    Human Immunology, 2018
    Co-Authors: Loes A. Van De Pasch, Maarten T. Penning, Karin Van Ham, Sanne Vendelbosch, Erik H. Rozemuller
    Abstract:

    Aim Killer-cell Immunoglobulin-like Receptors (KIRs) are encoded by 17 genes in the Leukocyte Receptor Complex (LRC). These genes are highly polymorphic, on the level of structural variants, and copy-number variation. These characteristics make it challenging to design specific amplifications, and to perform reliable analysis of each KIR allele present in a sample. To enable allele-level resolution KIR genotyping, we developed an NGS-based strategy that involves whole gene amplification of KIR genes. Methods We developed KIR-gene specific amplification primers, with specificity for one or more KIR genes (KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR3DS1, KIR3DL1, KIR3DL2, KIR3DL3 and KIR3DP1). Amplicons were processed in the NGSgo® (GenDx) library preparation procedure (GenDx) and paired-end sequenced on an Illumina MiSeq platform (2 × 151 bp). Data was genotyped with NGSengine® software (GenDx), using KIR database IPD-KIR 2.7.1. Results Multiple samples with known and unknown KIR genotypes were analyzed using this new strategy. KIR amplification, sequencing and subsequent typing was successful for the majority of samples. Many samples could be typed unambiguously, despite the fact that phasing was not fully accomplished due to the length of the genes (up to 17 kb). Remaining challenges in analysis are samples with KIR copy number variation and high degree of homology, which can hamper the data analysis. Conclusions KIR genotyping at allelic level is feasible using the short read sequencing technology of Illumina. This new typing strategy may be an attractive alternative to existing KIR genotype assays that only determine KIR gene content at a limited resolution, or complex sequencing methods aiming to sequence the entire LCR. L.A. van de Pasch: 5. Employee; Company/Organization; GenDx. K. van Ham: 5. Employee; Company/Organization; GenDx. S. Vendelbosch: 5. Employee; Company/Organization; GenDx. M.T. Penning: 5. Employee; Company/Organization; GenDx. E.H. Rozemuller: 5. Employee; Company/Organization; GenDx. 6. Stock Shareholder; Company/Organization; GenDx. 7. Other (Identify); Company/Organization; Founder of GenDx.

  • P101 Chimerism monitoring by next-generation sequencing: A multiplatform comparison
    Human Immunology, 2018
    Co-Authors: Evelien E. Bouwmans, Maarten T. Penning, Loes A. Van De Pasch, Erik H. Rozemuller
    Abstract:

    Aim Accurate monitoring of the chimeric status after stem cell transplantation is essential for early detection of relapse and at the moment mainly performed by STR or qPCR. The major disadvantages of STR are the laborious data analysis and poor sensitivity. The qPCR technique enables a much quicker workflow and better sensitivity. However a disadvantage is the need of a pre-transplant sample for each monitoring event. Performing chimerism monitoring by NGS will eliminate these limitations while allowing for a multiplexed setup, reducing the amount of needed lab work and DNA. The workflow can be combined with routinely applied NGS HLA typing in clinical laboratories. Methods PCR amplification primers have been designed for a set of biallelic markers. These markers were tested on a number of artificial chimeric samples in a range between 0.01–100% of positive DNA mixed with negative DNA. Amplicons were applied in the respective NGSgo library preparation workflows and run on Illumina MiSeq, Ion Torrent S5 and Oxford Nanopore MinION systems. NGS data was analysed with customized analysis tools designed to quantitate the two variants of each marker. Results Each marker was tested on a small panel of non-chimeric samples (Coriell Institute) and resulted in clear homo- or heterozygous patterns for all markers. Results of the different platforms on the artificial chimeric samples in a range of 0.01%–100% were compared. Sensitivity was determined for each sequencing platform. A read depth threshold was established for each of the platforms to achieve a minimum sensitivity of 0.05%. Results including difference between sequencing platforms will be discussed further. Conclusions The results demonstrate that NGS-based chimerism monitoring is feasible for a wide range of NGS platforms, with a high sensitivity and wide dynamic range. E.E. Bouwmans: 5. Employee; Company/Organization; GenDx. L.A. van de Pasch: 5. Employee; Company/Organization; GenDx. M.T. Penning: 5. Employee; Company/Organization; GenDx. E. Rozemuller: 5. Employee; Company/Organization; GenDx. 6. Stock Shareholder; Company/Organization; GenDx. 7. Other (Identify); Company/Organization; Founder of GenDx.

  • P092 Validation of the automated NGSGO library preparation workflow for HLA typing on the BIOMEK 4000
    Human Immunology, 2018
    Co-Authors: Bram Luiken, Maarten T. Penning, Debby Krom, Gayatri C. Tetar, Elena Tavkin, Shilpa Parakh, Alisa Jackson, Sake Van Wageningen, Wendy Swelsen
    Abstract:

    Aim The GenDx NGSgo workflow for HLA typing using Next-generation sequencing (NGS) consists of four practical steps: amplification, library preparation, sequencing and data analysis. Out of these four steps the library preparation is the most labor intensive and most interspersed by incubations. To decrease hands-on time and reduce the risk of human error, NGSgo library preparation can be automated from start to finish using a liquid handler workstation. Here we describe the results of the automation of the NGSgo library preparation for Illumina on a Beckman Coulter Biomek 4000 workstation. The workflow was fully automated from amplicon pool to finalized library, making it completely hands-off to accommodate overnight runs and allow the operator to step away from the machine. Methods The Biomek setup included a thermal cycler, 96-well plate magnet, two peltiers devices for cooling 96-well plates and 1.5 ml tubes, a gripper tool, reagent reservoirs and liquid waste disposal. Pipetting was performed by two pipet tools. This setup allowed for flexible processing of 4 to 24 samples per run, with any number of loci. Per run only three boxes of pipetting tips were used for 24 samples. Panels with variable numbers of samples and replicate panels were processed with the Biomek. Subsequently, the pooled libraries were sequenced on a MiSeq system and the resulting data was analyzed with NGSengine software (GenDx). Results MiSeq run parameters were indicative of a high-quality sequencing runs. Read depth, mappability and noise levels of the sequence data presented here showed that the automated workflow resulted in high quality reads and accurate typings. Results from the replicate panel showed a 100% typing match. Conclusions – NGSgo library preparation for 4 to 24 samples can be performed on the Biomek 4000 platform, without manual intervention. – The automated workflow results in high-quality reproducible libraries. – Performing automated NGSgo library preparation as described here reduces hands-on time from 1.5 h to 10 min as compared to manual execution. B. Luiken: 5. Employee; Company/Organization; GenDx. E. Tavkin: 5. Employee; Company/Organization; Beckman Coulter. S. Parakh: 5. Employee; Company/Organization; Beckman Coulter. J. Lu: 5. Employee; Company/Organization; Beckman Coulter. A. Jackson: 5. Employee; Company/Organization; Beckman Coulter. S. van Wageningen: 5. Employee; Company/Organization; GenDx. M. Penning: 5. Employee; Company/Organization; GenDx.

  • P080 HLA disease association assignment by multiplexed NGS assay
    Human Immunology, 2016
    Co-Authors: Nienke Westerink, Erik H. Rozemuller, Inge Van Rooy, Claudia Rebel, Michelle Van Heck, Maarten T. Penning
    Abstract:

    Aim Strong association of HLA molecules with autoimmune and inflammatory diseases have been identified, strengthening the value of HLA genetic screening for diagnostic purposes. There’s a strong association between Ankylosing Spondylitis (AS) and HLA-B27. In ∼90% of European patients, B27 alleles strongly predispose for AS. In ∼90% of patients with Coeliac Disease, the HLA-DQ2.5 phenotype is expressed, encoding HLA-DQA1∗05:01 and DQB1∗02:01 alleles, with the remaining 10% mostly expressing the HLA-DQ8 molecule, encoding the DQA1∗03 variant and DQB1∗03:02 alleles. Also in the field of pharmacogenetics there’s a growing interest in the role of HLA. HLA-B∗57:01 screening to prevent Abacavir hypersensitivity syndrome is now a routine clinical use in the developed world. We developed a novel NGS-based genetic screening test that enables 2nd field resolution typings of HLA-B, DQA1, and DQB1 in a single tube. Methods A multiplexed amplification assay was established that encompass HLA-B, HLA-DQA1 and HLA-DQB1 locus-specific amplification of exon 2 and 3 in a single tube. The assay was compatible with the multiplex PCR kit (Qiagen) and required a PCR enhancer (GenDx). Analytical performance studies were assessed including screening of a large gDNA reference panel (n = 96 samples) to verify robustness. Amplicons were pooled and processed into the NGSgo workflow for Illumina (GenDx) and analyzed by NGSengine software (GenDx). Results For all samples tested (n = 96) strong amplicons of the expected sizes were generated in a multiplexed PCR. NGS data showed high locus mappability (>94%), and full coverage of the amplicon with even distribution of reads, especially for HLA-B, and HLA-DQA1, having average read depths >1500. For all samples, HLA-B, HLA-DQB1 and HLA-DQA1 typings were obtained that are in concordance with the pre-types. The multiplexed assay showed to be robust and capable of detecting both alleles when present in a balanced manner. Conclusions We developed a novel NGS-based genetic screening test that enables HLA-B, DQA1, and DQB1 typings in a single tube. The genetic screening test proved to be robust and useful for pharmacogenetic screening of HLA-B alleles and identification of autoimmune disease associated genetic susceptibilities like celiac disease. N. Westerink:  Employee; Company/Organization; GenDx.I. van Rooy:  Employee; Company/Organization; Gendx. M. Van Heck:  Employee; Company/Organization; GenDx. C. Rebel:  Employee; Company/Organization; GenDx. E. Rozemuller:  Stock Shareholder; Company/Organization; GenDx. M. Penning:  Employee; Company/Organization; GenDx.

  • High-throughput and cost-effective NGS strategy for intermediate-resolution HLA typing
    Human Immunology, 2015
    Co-Authors: Inge Van Rooy, Michelle Bacelar, Erik H. Rozemuller, R Kooter, Claudia Rebel, Roel Van Aard, Bram Luiken, Loes A. Van De Pasch, Evelien E. Bouwmans, Maarten T. Penning
    Abstract:

    Aim The National Marrow Donor Program (NMDP) requires that each newly recruited donor is typed at intermediate resolution level for HLA-A, -B, -C, -DRB1 and optionally -DQB1. Therefore we developed an NGS assay specifically designed for intermediate resolution HLA typing (class I exon 2 & 3, and class II exon 2) that is suitable for high-throughput. PCR conditions were optimized such to reduce the costs of reagents. Moreover, the limited size of the amplicon allows for an increased sample capacity per run on either the MiSeq or Ion Torrent platform: a four-fold increase on average compared to the whole-gene amplification strategy. Method We integrated this NGSessenz strategy into the previously developed NGSgo workflows for MiSeq and Ion Torrent platforms. For the MiSeq platform, high-throughput and reproducibility was verified by running 4 × 96 DNA samples on a micro flow cell. For IonTorrent, we verified the capacity of the smallest (314 chip) and the largest (318 chip) by running a variety of samples. For each sample, loci were pooled prior to the execution of the NGSgo platform-specific library preparation and subsequent NGS procedures were performed. Results We will present the NGS data that were obtained for the robustness of amplification, mappability of the reads, read depth per locus, concordance of NGS typing with reference type, reproducibility of the NGS data generated, and the capacity of the NGSessenz assay on the different platforms. Conclusions Taken together, we will demonstrate the major advantage of this new NGSessenz strategy in ease-of-use and cost-effectiveness for HLA registry typing by means of NGS. I. Van Rooy: Employee; Company/Organization; GenDx. C. Rebel: Employee; Company/Organization; GenDx. R. Van Aard: Employee; Company/Organization; GenDx. M. Bacelar: Employee; Company/Organization; GenDx. B. Luiken: Employee; Company/Organization; GenDx. L. Van de Pasch: Employee; Company/Organization; GenDx. E. Bouwmans: Employee; Company/Organization; GenDx. R. Kooter: Employee; Company/Organization; GenDx. E. Rozemuller: Stock Shareholder; Company/Organization; GenDx. M. Penning: Employee; Company/Organization; GenDx. W. Mulder: Stock Shareholder; Company/Organization; GenDx. N. Westerink: Employee; Company/Organization; GenDx.

Nienke Westerink - One of the best experts on this subject based on the ideXlab platform.

  • P080 HLA disease association assignment by multiplexed NGS assay
    Human Immunology, 2016
    Co-Authors: Nienke Westerink, Erik H. Rozemuller, Inge Van Rooy, Claudia Rebel, Michelle Van Heck, Maarten T. Penning
    Abstract:

    Aim Strong association of HLA molecules with autoimmune and inflammatory diseases have been identified, strengthening the value of HLA genetic screening for diagnostic purposes. There’s a strong association between Ankylosing Spondylitis (AS) and HLA-B27. In ∼90% of European patients, B27 alleles strongly predispose for AS. In ∼90% of patients with Coeliac Disease, the HLA-DQ2.5 phenotype is expressed, encoding HLA-DQA1∗05:01 and DQB1∗02:01 alleles, with the remaining 10% mostly expressing the HLA-DQ8 molecule, encoding the DQA1∗03 variant and DQB1∗03:02 alleles. Also in the field of pharmacogenetics there’s a growing interest in the role of HLA. HLA-B∗57:01 screening to prevent Abacavir hypersensitivity syndrome is now a routine clinical use in the developed world. We developed a novel NGS-based genetic screening test that enables 2nd field resolution typings of HLA-B, DQA1, and DQB1 in a single tube. Methods A multiplexed amplification assay was established that encompass HLA-B, HLA-DQA1 and HLA-DQB1 locus-specific amplification of exon 2 and 3 in a single tube. The assay was compatible with the multiplex PCR kit (Qiagen) and required a PCR enhancer (GenDx). Analytical performance studies were assessed including screening of a large gDNA reference panel (n = 96 samples) to verify robustness. Amplicons were pooled and processed into the NGSgo workflow for Illumina (GenDx) and analyzed by NGSengine software (GenDx). Results For all samples tested (n = 96) strong amplicons of the expected sizes were generated in a multiplexed PCR. NGS data showed high locus mappability (>94%), and full coverage of the amplicon with even distribution of reads, especially for HLA-B, and HLA-DQA1, having average read depths >1500. For all samples, HLA-B, HLA-DQB1 and HLA-DQA1 typings were obtained that are in concordance with the pre-types. The multiplexed assay showed to be robust and capable of detecting both alleles when present in a balanced manner. Conclusions We developed a novel NGS-based genetic screening test that enables HLA-B, DQA1, and DQB1 typings in a single tube. The genetic screening test proved to be robust and useful for pharmacogenetic screening of HLA-B alleles and identification of autoimmune disease associated genetic susceptibilities like celiac disease. N. Westerink:  Employee; Company/Organization; GenDx.I. van Rooy:  Employee; Company/Organization; Gendx. M. Van Heck:  Employee; Company/Organization; GenDx. C. Rebel:  Employee; Company/Organization; GenDx. E. Rozemuller:  Stock Shareholder; Company/Organization; GenDx. M. Penning:  Employee; Company/Organization; GenDx.

  • High-resolution HLA typings obtained for high-throughput NGS using the Illumina MiSeq platform
    Human Immunology, 2015
    Co-Authors: Loes A. Van De Pasch, Michelle Bacelar, Maarten T. Penning, Erik H. Rozemuller, R Kooter, Claudia Rebel, Roel Van Aard, Evelien E. Bouwmans, Nienke Westerink
    Abstract:

    Aim Next-generation sequencing (NGS) technology generates sequences from single DNA molecules that enables unique identification of the paternal and maternal alleles. The NGS technology has great potential in the application of HLA typing for diagnostic purposes and generating reliable, unambiguous HLA typing results in a high-throughput fashion. Here we present data obtained for 11 HLA loci using a HLA typing assay dedicated for high-throughput NGS-based HLA typing on the Illumina MiSeq. The implementation of this high-throughput NGS strategy would be highly advantageous to obtain robust high-resolution HLA typing results for registry labs and routine diagnostic purposes. Method A large genomic DNA reference panel, including UCLA reference samples, were selected for high-throughput NGS HLA typing. The whole gene of HLA-A, -B, -C, and the essential genomic regions of HLA-DRB1, -DRB345, -DQA1, -DQB1, -DPA1 and -DPB1 were PCR-amplified in a single PCR for each locus. Amplicons of each sample were pooled and processed using our NGSgo® DNA library preparation workflow for Illumina MiSeq. During library preparation the samples were fragmented, barcoded and multiplexed. Finally paired-end sequencing (2x150 cycles) was performed on the Illumina MiSeq platform using a standard flow cell and data was analysed using our HLA typing software NGSengine®. Results Paired-end NGS data was obtained for 11 HLA loci (class I and II) of most genomic DNA reference samples in a single Illumina MiSeq sequencing run. The NGS data covered the complete HLA region of interest by highly accurate measures for all HLA loci tested. All HLA alleles were effectively separated and, in most cases, could be phased throughout the gene. For the majority of samples, unambiguous third- and fourth-field resolution typing were obtained for all loci that are in concordance with the second-field or higher reference types. Conclusions The high-throughput NGSgo® strategy that we have developed demonstrates to be a powerful method to perform high-resolution HLA typing by means of NGS in a robust and reliable manner. L. Van de Pasch: Employee; Company/Organization; GenDx. M. Bacelar: Employee; Company/Organization; GenDx. C. Rebel: Employee; Company/Organization; GenDx. R. Van Aard: Employee; Company/Organization; GenDx. R. Kooter: Employee; Company/Organization; GenDx. E. Rozemuller: Stock Shareholder; Company/Organization; GenDx. M. Penning: Employee; Company/Organization; GenDx. E. Bouwmans: Employee; Company/Organization; GenDx. N. Westerink: Employee; Company/Organization; GenDx.

  • Rapid microchimerism analysis using multiplexed reagents and novel software
    Human Immunology, 2015
    Co-Authors: Bram Luiken, Nienke Westerink, Erik H. Rozemuller, Job Geerlings, Doug Bost
    Abstract:

    Aim To overcome the sensitivity and analysis shortcomings of STR-based transplant monitoring methods, and to improve upon the current state of the art in qPCR-based chimerism analysis, KimerDx has developed a new assay and software suite. Method Our reagents employ a multiplexed panel of 30 qPCR research assays and software to genotype multiple samples on a single plate and identify informative markers. The software presents the marker choices in their genomic context, allowing for informed decision-making and use in post-transplant monitoring. The genotyping results are stored by the software for recall during subsequent monitoring. Results Our software facilitates post-transplant monitoring by: allowing operators to customize their plate configuration or work with a lab-defined template, performing all calculations necessary to execute the test, generating a printable protocol to assist laboratory work and by generating templates to import into the qPCR machines. The software accepts standard qPCR data output for generating results. Reports from a monitoring test may be generated from a single time point or using the longitudinal data collected from the individual sample over time – providing a temporal view for better understanding rejection or relapse kinetics. Conclusion Our reagents and software are compatible with qPCR platforms from multiple vendors. The software solution reduces manual calculations, increases flexibility in experimental execution, generates complete protocols for use in the lab and stores results. B. Luiken: Employee; Company/Organization; GenDx. J. Geerlings: Employee; Company/Organization; GenDx. N. Westerink: Employee; Company/Organization; GenDx. E. Rozemuller: Stock Shareholder; Company/Organization; GenDx, KimerDx. D. Bost: Employee; Company/Organization; KimerDx BV. 6. Stock Shareholder; Company/Organization; KimerDx BV.

  • P094 : A FULL NGS WORKFLOW FOR REACHING THE ULTIMATE HLA TYPING RESOLUTION
    Human Immunology, 2014
    Co-Authors: J. Adema, Michelle Bacelar, Frans Paul Ruzius, Maarten T. Penning, Erik H. Rozemuller, R Kooter, Loes A. Van De Pasch, Nienke Westerink
    Abstract:

    Aim Currently applied technologies for HLA sequencing-based typing (SBT) often show ambiguous results, most of which are caused by cis–trans allele combinations. In contrast, next-generation sequencing (NGS) allows for reads that originate from a single molecule, meaning that the paternal and maternal alleles can uniquely be identified. As NGS can phase single molecules throughout the gene, HLA typing by means of NGS will yield typing results without ambiguities. The implementation of NGS technology for routine diagnostic purposes would therefore be highly advantageous to obtain reliable, unambiguous HLA typing results. Methods We developed a complete NGS workflow for high-throughput HLA typing compatible with the Illumina NGS platforms. The NGS workflow, including full-length HLA locus amplification, library preparation, and sequencing analysis, is fully optimized to facilitate high-resolution identification of HLA alleles. We generated HLA locus specific amplicons for the class I loci (HLA-A, -B and -C) and class II loci (HLA-DRB1, -DRB3, -DRB4, -DRB5, -DPA1, -DPB1, -DQA1, and -DQB1) on 96 genomic DNA reference samples, using our optimized NGS workflow compatible with the Illumina MiSeq platform. Samples and loci were pooled and run on the MiSeq. Sequence data was analyzed using the NGS HLA typing software NGSengine®. Results Paired-end NGS data was obtained for 11 class I and class II HLA loci pooled for 96 gDNA reference samples in one MiSeq sequencing run. The NGS data covered the complete HLA region of interest by highly accurate measures for all HLA loci tested. All HLA alleles were effectively separated and, in most cases, could be phased throughout the gene, resulting in unambiguous high-resolution HLA typings. High-quality data was obtained for all loci in agreement with the reference SBT allele assignments as provided by Sanger SBT. Conclusion The NGS workflow we have developed demonstrates that it is feasible to perform HLA typing by means of NGS in a reliable, accurate and high-throughput manner that in future will be suitable for routine diagnostic purposes. J. Adema: Employee; Company/Organization; GenDx. M. Bacelar: Employee; Company/Organization; GenDx. R. Kooter: Employee; Company/Organization; GenDx. F. Ruzius: Employee; Company/Organization; GenDx. E.H. Rozemuller: Employee; Company/Organization; GenDx. Stock Shareholder; Company/Organization; GenDx. M.T. Penning: Employee; Company/Organization; GenDx. L. van de Pasch: Employee; Company/Organization; GenDx. N. Westerink: Employee; Company/Organization; GenDx.

  • P097 : ACCURATE HLA TYPING BY NGS USING THE IonTorrent PGM WITH A PLATFORM SPECIFIC DEVELOPED AND TESTED WORKFLOW
    Human Immunology, 2014
    Co-Authors: Laura Krol, Frans Paul Ruzius, Erik H. Rozemuller, R Kooter, J. Adema, Loes A. Van De Pasch, Nienke Westerink
    Abstract:

    Aim Next-generation sequencing (NGS) technology has great potential for the future application of HLA typing in routine diagnostic purposes. NGS sequencing has the advantage that it allows the sequencing of single molecule DNA sequences, meaning that the paternal and maternal alleles can be uniquely identified. As NGS can phase single molecules throughout the gene, HLA typing by means of NGS will yield typing results without ambiguities. Currently, various NGS platforms are available on the market; each platform is based on different technology and each technology has their own specific requirements in order to make them compatible for HLA typing. It is our aim to develop multiple NGS workflows that are each compatible to a specific NGS platform, such as the Illumina MiSeq, the IonTorrent PGM, and the PacBio RS Sequencer. Method Here we present our latest developments on the IonTorrent PGM platform specific workflow, which is designed for the high-resolution typing of HLA loci. The new IonTorrent workflow for HLA typing includes several consecutive steps, from the most crucial step of whole HLA locus amplification to fragmentation, adaptor ligation, suppression PCR, clonal amplification by emulsion PCR, enrichment of positive ion sphere particles and sequencing on the IonTorrent PGM platform. Results We present the application of the IonTorrent workflow for 24 samples from standardized genomic DNA reference panels. GenDx adapters are developed and applied, enabling indexing of multiple samples. The NGS data is analyzed using the NGS platform-independent NGSengine® software, developed for NGS HLA sequence analysis (GenDx). Conclusion The availability of different NGS workflows allows the end-user to choose most preferred SBT-NGS HLA typing method that fulfills their laboratory application needs and requirements. This GenDx NGS workflow for the IonTorrent PGM presented here, is a powerful method to perform HLA typing by means of NGS in a reliable, accurate and high-throughput manner. L. Krol: Employee; Company/Organization; GenDx. J. Adema: Employee; Company/Organization; GenDx. R. Kooter: Employee; Company/Organization; GenDx. F. Ruzius: Employee; Company/Organization; GenDx. E.H. Rozemuller: Employee; Company/Organization; GenDx. Stock Shareholder; Company/Organization; GenDx. L. van de Pasch: Employee; Company/Organization; GenDx. N. Westerink: Employee; Company/Organization; GenDx.

Anajane G. Smith - One of the best experts on this subject based on the ideXlab platform.

  • p099 comparison of ssop versus ngs for typing of hla a b c drb1 drb3 b4 b5 dqa1 dqb1 dpa1 dpb1 toward single pass high resolution hla typing in support of solid organ and hematopoietic cell transplant programs
    Human Immunology, 2018
    Co-Authors: Anajane G. Smith, Shalini Pereira, Wyatt Nelson, Andres Jaramillo, Faisal Khan, Noureddine Berka, Marcelo J Pando, Maria Bettinotti, Medhat Askar
    Abstract:

    Aim Many laboratories use a 2-tier HLA typing process: a first pass intermediate resolution and then high resolution, when clinically necessary. SSP or qPCR provide rapid typing for deceased donor workup; SSOP is widely employed for higher volumes; and Sanger sequencing has been the gold standard for high resolution. However, SSOP and Sanger SBT often yield ambiguous results and resolution is expensive and time consuming. Next generation sequencing offers a single pass technology to achieve unambiguous HLA allele assignments. We compared commercial SSOP and NGS systems with respect to accuracy, turnaround time, effort and resolution level. Methods Five laboratories submitted coded, blinded samples, previously typed by SSOP at each institute, to a separate lab for NGS of HLA-A, B, C at exons 1–7, DRB1, DRB3/4/5, DQA1, DQB1, DPB1 at exons 1–4, and DPA1 at exons 2–4. For up to 24 samples by SSOP or up to 48 samples by NGS, benchwork is completed by one technologist on day 1 with results available for reporting on day 2. Results At the SSOP resolution level, results were concordant except for 11 SSOP assignments in 8 specimens due to false probe reactions at DRB1, DRB5, DQA1, DQB1, and DPB1. NGS identified 21 novel sequences: one with a multi-exon deletion and 20 with SNP polymorphisms in HLA-C, DPA1, DPB1, DQA1, DRB1, DRB3, DRB4, and DRB5 alleles. Across the highly polymorphic HLA-A, B, C, DRB1 loci, only 1% of intermediate resolution, IR, SSOP results were unambiguous. Even higher resolution, HR, SSOP by 1 lab gave only 17% specific allele assignments. In contrast, over 99% of NGS results were specific unambiguous genotypes. Across all loci, NGS typing was specific except for certain discrete diploid ambiguities, also found in SSOP. Conclusions This study provides a compelling rationale for implementing NGS for single pass HLA analysis with accurate, 2 day turnaround, which includes the high resolution, unambiguous genotyping critical for unrelated donor HCT and that may be required for sensitized patients with allele specific antibodies. A.G. Smith: 2. Consultant; Company/Organization; Scisco Genetics Inc. S.E. Pereira: 4. Scientific/Medical Advisor; Company/Organization; Scisco Genetics Inc. C. Pyo: 4. Scientific/Medical Advisor; Company/Organization; Scisco Genetics Inc. W. Nelson: 5. Employee; Company/Organization; Scisco Genetics Inc. M. Bettinotti: 3. Speaker’s Bureau; Company/Organization; One Lambda ThermoFisher. M.Z. Askar: 3. Speaker’s Bureau; Company/Organization; Immucor. 4. Scientific/Medical Advisor; Company/Organization; Illumina. D.E. Geraghty: 6. Stock Shareholder; Company/Organization; Scisco Genetics Inc. Download high-res image (283KB) Download full-size image

  • OR27. Novel next generation sequencing based chimerism assay for engraftment monitoring in hematopoietic cell transplantation
    Human Immunology, 2018
    Co-Authors: Medhat Askar, Anajane G. Smith, Shalini Pereira, Chul-woo Pyo, Amanda Willis, Jenifer D. Williams, Leah Pittmon, Wyatt C. Nelson, Daniel E. Geraghty
    Abstract:

    Aim Most laboratories providing chimerism testing use electrophoretic analysis of STR loci or qPCR of SNP polymorphisms. STR assays are limited to 1–5% sensitivity and qPCR requires substantial amounts of DNA which may not be feasible in patients with low blood cell counts or for analysis of rare cell subsets. We developed a chimerism assay based on NGS of SNP and InDel polymorphisms and validated it in a clinical transplant immunology lab supporting a large HCT program. Methods Multiple polymorphic loci were evaluated for specificity, sensitivity and accuracy using artificial admixtures as well as direct comparison to qPCR results from patient specimens post HCT. An initial panel comprised 37 SNPs on 18 chromosomes and 12 InDels on 10 chromosomes. Target generation is performed in 2 multiplexed PCRs with 5 ng sample DNA per reaction. All NGS benchwork is accomplished within 6 h, followed by a 7 h MiSeq run, so that results are available for reporting early on day 2. Results Initial NGS testing used artificial admixtures of unrelated individuals, with mixtures from 10% to 0.1%, see Table 1. On average, 30 informative markers were identified between unrelated individuals and NGS achieved 0.5% sensitivity among all 4 admixture sets. Table 2 shows the results for NGS versus qPCR for 7 patient post HCT specimens. Download : Download high-res image (334KB) Download : Download full-size image Download : Download high-res image (247KB) Download : Download full-size image Conclusions These preliminary results show that NGS technology can provide accurate, informative, and sensitive detection of a minority species with minimal DNA quantity requirements, rapid turnaround and minimal hands on time. As laboratories implement NGS technology for high resolution genotyping of HLA and other genetic loci, extension to engraftment monitoring might also offer workplace efficiencies. M.Z. Askar: 3. Speaker’s Bureau; Company/Organization; Immucor. 4. Scientific/Medical Advisor; Company/Organization; Illumina. S.E. Pereira: 4. Scientific/Medical Advisor; Company/Organization; Scisco Genetics Inc. A.G. Smith: 2. Consultant; Company/Organization; Scisco Genetics Inc. C. Pyo: 4. Scientific/Medical Advisor; Company/Organization; Scisco Genetics Inc. W. Nelson: 5. Employee; Company/Organization; Scisco Genetics Inc. D.E. Geraghty: 6. Stock Shareholder; Company/Organization; Scisco Genetics Inc.

  • P099 Comparison of ssop versus ngs for typing of HLA-A, B, C, DRB1, DRB3/B4/B5, DQA1, DQB1, DPA1, DPB1: Toward single pass high resolution hla typing in support of solid organ and hematopoietic cell transplant programs
    Human Immunology, 2018
    Co-Authors: Anajane G. Smith, Shalini Pereira, Andres Jaramillo, Faisal Khan, Noureddine Berka, Marcelo J Pando, Maria Bettinotti, Chul-woo Pyo, Wyatt C. Nelson, Medhat Askar
    Abstract:

    Aim Many laboratories use a 2-tier HLA typing process: a first pass intermediate resolution and then high resolution, when clinically necessary. SSP or qPCR provide rapid typing for deceased donor workup; SSOP is widely employed for higher volumes; and Sanger sequencing has been the gold standard for high resolution. However, SSOP and Sanger SBT often yield ambiguous results and resolution is expensive and time consuming. Next generation sequencing offers a single pass technology to achieve unambiguous HLA allele assignments. We compared commercial SSOP and NGS systems with respect to accuracy, turnaround time, effort and resolution level. Methods Five laboratories submitted coded, blinded samples, previously typed by SSOP at each institute, to a separate lab for NGS of HLA-A, B, C at exons 1–7, DRB1, DRB3/4/5, DQA1, DQB1, DPB1 at exons 1–4, and DPA1 at exons 2–4. For up to 24 samples by SSOP or up to 48 samples by NGS, benchwork is completed by one technologist on day 1 with results available for reporting on day 2. Results At the SSOP resolution level, results were concordant except for 11 SSOP assignments in 8 specimens due to false probe reactions at DRB1, DRB5, DQA1, DQB1, and DPB1. NGS identified 21 novel sequences: one with a multi-exon deletion and 20 with SNP polymorphisms in HLA-C, DPA1, DPB1, DQA1, DRB1, DRB3, DRB4, and DRB5 alleles. Across the highly polymorphic HLA-A, B, C, DRB1 loci, only 1% of intermediate resolution, IR, SSOP results were unambiguous. Even higher resolution, HR, SSOP by 1 lab gave only 17% specific allele assignments. In contrast, over 99% of NGS results were specific unambiguous genotypes. Across all loci, NGS typing was specific except for certain discrete diploid ambiguities, also found in SSOP. Conclusions This study provides a compelling rationale for implementing NGS for single pass HLA analysis with accurate, 2 day turnaround, which includes the high resolution, unambiguous genotyping critical for unrelated donor HCT and that may be required for sensitized patients with allele specific antibodies. A.G. Smith: 2. Consultant; Company/Organization; Scisco Genetics Inc. S.E. Pereira: 4. Scientific/Medical Advisor; Company/Organization; Scisco Genetics Inc. C. Pyo: 4. Scientific/Medical Advisor; Company/Organization; Scisco Genetics Inc. W. Nelson: 5. Employee; Company/Organization; Scisco Genetics Inc. M. Bettinotti: 3. Speaker’s Bureau; Company/Organization; One Lambda ThermoFisher. M.Z. Askar: 3. Speaker’s Bureau; Company/Organization; Immucor. 4. Scientific/Medical Advisor; Company/Organization; Illumina. D.E. Geraghty: 6. Stock Shareholder; Company/Organization; Scisco Genetics Inc. Download : Download high-res image (283KB) Download : Download full-size image

  • OR47 The mystery of the missing DRB1 allele – solved by NGS!!!
    Human Immunology, 2018
    Co-Authors: Anajane G. Smith, Shalini Pereira, Medhat Askar, Chul-woo Pyo, Amanda Willis, Wyatt C. Nelson, Daniel E. Geraghty
    Abstract:

    The mystery began when HLA typing, of two apparently unrelated individuals at two different institutions, identified the presence of a DRB5∗01 gene without any evidence of an expected linked DRB1∗15, DRB1∗16, or, in rare cases, DRB1∗01 allele. Historic data from Sanger sequence-based typing included DRB1∗14, DRB3∗02:02, DRB5∗01:01, DQB1∗05:03, 06:02 for Individual 1 and DRB1∗04:01; DRB4∗01; DRB5∗01:01; DQB1∗03:01, 06:02 for Individual 2. Since those analyses only assessed exon 2, the apparent absence of a DRB1 allele associated with the DRB5 gene raised the possibility of an entire linked DRB1 gene having gone missing. Both specimens were recently re-examined using next generation sequencing technology. HLA-A, B, C genes were assessed for exons 1–7; DRB1, DRB3/B4/B5, DQA1, DQB1, and DPB1 were assessed for exons 1–4; and DPA1 for exons 2–4. In Individual 1, NGS identified DRB1∗14:54:01 and a DRB1∗15 exon 1 sequence, but without any evidence of DRB1∗15 sequences for exons 2 and 3. Since the exon 4 sequences of DRB1∗14:54:01 and DRB1∗15 are identical, it could not be determined whether the DRB1∗15 exon 4 sequence was present. Individual 2, typed as DRB1∗04:01:01 and, again, a DRB1∗15 exon 1. In the latter case, it was clear that no exon 2, 3, or 4 sequences were present for the DRB1∗15 gene, since exon 4 sequences differ between DRB1∗04:01:01 and DRB1∗15. The table shows the full NGS typing data. The HLA alleles shared by both individuals suggest a putative extended haplotype: A∗25:01:01-C∗12:03:01-B∗18:01:01-DRB1∗15new-DRB5∗01:01:01-DQA1∗01:02:01-DQB1∗06:02:01-DPA1∗01:03:01-DPB1∗23:01:01. NGS typing solved the missing allele mystery through identification of a truncated DRB1∗15 allele comprising an exon 1 sequence but without exons 2, 3, or 4. Similarly, there is DRB4∗03:01N which has an exon 3 sequence of a DRB4∗01:01, but without an exon 2 sequence. These unusual alleles appear to be the results of historic multi-exon deletion events and add to the astounding diversity of the human major histocompatibility complex. Download high-res image (242KB) Download full-size image A.G. Smith: 2. Consultant; Company/Organization; Scisco Genetics Inc. S.E. Pereira: 4. Scientific/Medical Advisor; Company/Organization; Scisco Genetics Inc. C. Pyo: 4. Scientific/Medical Advisor; Company/Organization; Scisco Genetics Inc. W. Nelson: 5. Employee; Company/Organization; Scisco Genetics Inc. M.Z. Askar: 3. Speaker’s Bureau; Company/Organization; Immucor. 4. Scientific/Medical Advisor; Company/Organization; Illumina. D.E. Geraghty: 6. Stock Shareholder; Company/Organization; Scisco Genetics Inc.