- Email: [email protected]
Rapid microchimerism analysis using multiplexed reagents and novel software
88
P072
Abstracts / Human Immunology 76 (2015) 38–167
HIGH-RESOLUTION HLA TYPINGS OBTAINED FOR HIGH-THROUGHPUT NGS USING THE ILLUMINA MISEQ PLATFORM. Loes Van de Pasch, Michelle Bacelar, Claudia Rebel, Roel Van Aard, Raul Kooter, Erik Rozemuller, Maarten Penning, Evelien Bouwmans, Nienke Westerink. GenDx, Utrecht, Netherlands. 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 NGSgoÒ 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 NGSengineÒ. 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 NGSgoÒ 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.
P073
RAPID MICROCHIMERISM ANALYSIS USING MULTIPLEXED REAGENTS AND NOVEL SOFTWARE. Bram Luiken a, Job Geerlings a, Nienke Westerink a, Erik Rozemuller a, Doug Bost b. a GenDx, Utrecht, Netherlands; b KimerDx BV, Utrecht, Netherlands. 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.
P072
Abstracts / Human Immunology 76 (2015) 38–167
HIGH-RESOLUTION HLA TYPINGS OBTAINED FOR HIGH-THROUGHPUT NGS USING THE ILLUMINA MISEQ PLATFORM. Loes Van de Pasch, Michelle Bacelar, Claudia Rebel, Roel Van Aard, Raul Kooter, Erik Rozemuller, Maarten Penning, Evelien Bouwmans, Nienke Westerink. GenDx, Utrecht, Netherlands. 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 NGSgoÒ 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 NGSengineÒ. 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 NGSgoÒ 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.
P073
RAPID MICROCHIMERISM ANALYSIS USING MULTIPLEXED REAGENTS AND NOVEL SOFTWARE. Bram Luiken a, Job Geerlings a, Nienke Westerink a, Erik Rozemuller a, Doug Bost b. a GenDx, Utrecht, Netherlands; b KimerDx BV, Utrecht, Netherlands. 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.