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Generation of high confidence HLA genotyping and consensus sequences for class I HLA loci using the NGS-based Omixon Holotype HLA typing system and the Illumina Miseq platform
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P130
Abstracts / Human Immunology 76 (2015) 38–167
VALIDATION OF A CLINICAL ASSAY FOR SPECIFIC GENOTYPING OF A KIR2DL1 ALLELIC POLYMORPHISM. Paula Arnold, Po-Chien Chou, Rafijul Bari, Wing Leung. St. Jude Children’s Research Hospital, Memphis, TN, United States. Aim: A number of transplant centers currently use killer-cell immunoglobulin-like receptor (KIR) genotyping as criteria for donor selection for hematopoietic stem cell transplant (HSCT) and natural killer (NK) cell protocols. Several factors contribute to donor selection, including presence or absence of multiple inhibitory and stimulatory KIR, centromeric vs. telomeric KIR haplotypes, ‘‘A’’ vs. ‘‘B’’ content KIR haplotypes, and NK cell education or licensing. Recent studies at our institution demonstrated that amino acid residue 245 of the KIR2DL1 protein is important in determining the signaling capacity, and had a significant effect on the overall outcome of HSCT. Patients who received a donor graft containing KIR2DL1 alleles encoding an arginine at position 245 (KIR2DL1-R245) had better survival and lower incidence of disease progression than the patients who received a donor graft containing KIR2DL1 alleles encoding a cysteine at the same position (KIR2DL1-C245). The goal of this work was to validate a lab-developed clinical test to distinguish the KIR2DL1 R245/C245 allelic polymorphism and to facilitate donor selection based on this criterion. Methods: Our research laboratory developed a real-time PCR single nucleotide polymorphism (SNP) assay that specifically detects alleles of the KIR2DL1 gene encoding arginine or cysteine at position 245, and avoids the interference of 2DL2 alleles that share the same sequence flanking this region. The HLA laboratory used the backbone of this assay to validate it for clinical use as a lab-developed companion test to current KIR genotyping. Results: The SNP assay performed according to the required qualifications, including accuracy, precision, sensitivity, and specificity. Samples tested showed 100% concordance with the expected genotyping of the ‘‘Gold Standard,’’ cell lines with sequenced KIR2DL1 alleles from the CIBMTR Repository. Conclusions: This SNP assay was validated for clinical use in the HLA laboratory, and should be in use in the near future as a companion test to the KIR genotyping currently used for donor selection in HSCT. P. Arnold: Grant/Research Support; Company/Organization; American Lebanese Associated Charities (ALSAC). P. Chou: Grant/Research Support; Company/Organization; American Lebanese Syrian Associated Charities (ALSAC). R. Bari: Grant/Research Support; Company/Organization; American Lebanese Syrian Associated Charities (ALSAC), Assisi Foundation of Memphis. 7. Other (Identify); Company/Organization; Dr. Bari is named as an inventor on patent applications claiming a SNP assay used for KIR allele typing, which is owned by SJCRH and is licensed to a commercial entity. W. Leung: Grant/Research Support; Company/Organization; American Lebanese Syrian Associated Charities (ALSAC), Assisi Foundation of Memphis. 7. Other (Identify); Company/Organization; Dr. Leung is named as an inventor on patent applications claiming a SNP assay used for KIR allele typing, which is owned by SJCRH and is licensed to a commercial entity.
P131
GENERATION OF HIGH CONFIDENCE HLA GENOTYPING AND CONSENSUS SEQUENCES FOR CLASS I HLA LOCI USING THE NGS-BASED OMIXON HOLOTYPE HLA TYPING SYSTEM AND THE ILLUMINA MISEQ PLATFORM. Peter M. Clark, Jamie Duke, Deborah Ferriola, Anna Papazoglou, Aniqa Hassan, Dimitri Monos. The Children’s Hospital of Philadelphia, Philadelphia, PA, United States. Aim: Generate HLA genotyping and high confidence consensus sequences for 50 samples at HLA-A, B and C loci using the Omixon Holotype HLA typing system. Methods: The Omixon Holotype HLA typing kit was used to generate sequencing libraries for 50 samples for HLA-A, B and C loci, and sequenced on an Illumina MiSeq. HLA typing and consensus sequence generation was performed using Omixon Twin, GenDx NGSengine and a custom pipeline, which relies on stringently mapping reads to the genotyped alleles and using SAMtools to generate the consensus. Consensus sequences generated by each method were aligned pairwise to determine concordance using a semi-global Needleman– Wunsch algorithm. When the sequences generated by each method were concordant, sequences were aligned to the IMGT reference sequence of the genotyped allele, to annotate novel alleles and annotate the sequence of incomplete alleles. When sequences generated by each method were discordant, sequences were aligned to the genotyped allele for manual inspection and assessment of algorithm performance. Results: HLA genotyping results for all 300 alleles (50 samples at 3 loci) were 100% concordant between NGSengine and Omixon Twin, after manually resolving discrepancies (6/300). Consensus sequences were generated for every allele (300) encompassing 31 unique HLA-A, 52 unique HLA-B and 31 unique HLA-C alleles. Our analysis facilitated the annotation of full gene sequences for 11 HLA alleles for which there is no complete IMGT reference sequence (A⁄02:04, B⁄14:03, B⁄15:08:01, B⁄15:30, B⁄27:02:01, B⁄35:04:01, B⁄35:43:01, B⁄39:09:01, B⁄51:08:01, B⁄58:02 and C⁄07:18) and facilitated the discovery of two novel HLA-A alleles, with intronic substitutions. For the remaining alleles, consensus sequences were compared against the reference sequences of the genotyped alleles and occasional differences between consensus sequences and reference sequences were manually inspected to evaluate algorithm performance. It has been determined that depth of coverage is critical in phasing and base calling, which is necessary to generate credible consensus sequences. Conclusions: Generation of high confidence HLA class I consensus sequences is possible using the Omixon Holotype HLA typing system provided that adequate depth of uniform coverage is obtained from sequencing. D. Monos: Other (Identify); Company/Organization; Omixon Royalties.
P130
Abstracts / Human Immunology 76 (2015) 38–167
VALIDATION OF A CLINICAL ASSAY FOR SPECIFIC GENOTYPING OF A KIR2DL1 ALLELIC POLYMORPHISM. Paula Arnold, Po-Chien Chou, Rafijul Bari, Wing Leung. St. Jude Children’s Research Hospital, Memphis, TN, United States. Aim: A number of transplant centers currently use killer-cell immunoglobulin-like receptor (KIR) genotyping as criteria for donor selection for hematopoietic stem cell transplant (HSCT) and natural killer (NK) cell protocols. Several factors contribute to donor selection, including presence or absence of multiple inhibitory and stimulatory KIR, centromeric vs. telomeric KIR haplotypes, ‘‘A’’ vs. ‘‘B’’ content KIR haplotypes, and NK cell education or licensing. Recent studies at our institution demonstrated that amino acid residue 245 of the KIR2DL1 protein is important in determining the signaling capacity, and had a significant effect on the overall outcome of HSCT. Patients who received a donor graft containing KIR2DL1 alleles encoding an arginine at position 245 (KIR2DL1-R245) had better survival and lower incidence of disease progression than the patients who received a donor graft containing KIR2DL1 alleles encoding a cysteine at the same position (KIR2DL1-C245). The goal of this work was to validate a lab-developed clinical test to distinguish the KIR2DL1 R245/C245 allelic polymorphism and to facilitate donor selection based on this criterion. Methods: Our research laboratory developed a real-time PCR single nucleotide polymorphism (SNP) assay that specifically detects alleles of the KIR2DL1 gene encoding arginine or cysteine at position 245, and avoids the interference of 2DL2 alleles that share the same sequence flanking this region. The HLA laboratory used the backbone of this assay to validate it for clinical use as a lab-developed companion test to current KIR genotyping. Results: The SNP assay performed according to the required qualifications, including accuracy, precision, sensitivity, and specificity. Samples tested showed 100% concordance with the expected genotyping of the ‘‘Gold Standard,’’ cell lines with sequenced KIR2DL1 alleles from the CIBMTR Repository. Conclusions: This SNP assay was validated for clinical use in the HLA laboratory, and should be in use in the near future as a companion test to the KIR genotyping currently used for donor selection in HSCT. P. Arnold: Grant/Research Support; Company/Organization; American Lebanese Associated Charities (ALSAC). P. Chou: Grant/Research Support; Company/Organization; American Lebanese Syrian Associated Charities (ALSAC). R. Bari: Grant/Research Support; Company/Organization; American Lebanese Syrian Associated Charities (ALSAC), Assisi Foundation of Memphis. 7. Other (Identify); Company/Organization; Dr. Bari is named as an inventor on patent applications claiming a SNP assay used for KIR allele typing, which is owned by SJCRH and is licensed to a commercial entity. W. Leung: Grant/Research Support; Company/Organization; American Lebanese Syrian Associated Charities (ALSAC), Assisi Foundation of Memphis. 7. Other (Identify); Company/Organization; Dr. Leung is named as an inventor on patent applications claiming a SNP assay used for KIR allele typing, which is owned by SJCRH and is licensed to a commercial entity.
P131
GENERATION OF HIGH CONFIDENCE HLA GENOTYPING AND CONSENSUS SEQUENCES FOR CLASS I HLA LOCI USING THE NGS-BASED OMIXON HOLOTYPE HLA TYPING SYSTEM AND THE ILLUMINA MISEQ PLATFORM. Peter M. Clark, Jamie Duke, Deborah Ferriola, Anna Papazoglou, Aniqa Hassan, Dimitri Monos. The Children’s Hospital of Philadelphia, Philadelphia, PA, United States. Aim: Generate HLA genotyping and high confidence consensus sequences for 50 samples at HLA-A, B and C loci using the Omixon Holotype HLA typing system. Methods: The Omixon Holotype HLA typing kit was used to generate sequencing libraries for 50 samples for HLA-A, B and C loci, and sequenced on an Illumina MiSeq. HLA typing and consensus sequence generation was performed using Omixon Twin, GenDx NGSengine and a custom pipeline, which relies on stringently mapping reads to the genotyped alleles and using SAMtools to generate the consensus. Consensus sequences generated by each method were aligned pairwise to determine concordance using a semi-global Needleman– Wunsch algorithm. When the sequences generated by each method were concordant, sequences were aligned to the IMGT reference sequence of the genotyped allele, to annotate novel alleles and annotate the sequence of incomplete alleles. When sequences generated by each method were discordant, sequences were aligned to the genotyped allele for manual inspection and assessment of algorithm performance. Results: HLA genotyping results for all 300 alleles (50 samples at 3 loci) were 100% concordant between NGSengine and Omixon Twin, after manually resolving discrepancies (6/300). Consensus sequences were generated for every allele (300) encompassing 31 unique HLA-A, 52 unique HLA-B and 31 unique HLA-C alleles. Our analysis facilitated the annotation of full gene sequences for 11 HLA alleles for which there is no complete IMGT reference sequence (A⁄02:04, B⁄14:03, B⁄15:08:01, B⁄15:30, B⁄27:02:01, B⁄35:04:01, B⁄35:43:01, B⁄39:09:01, B⁄51:08:01, B⁄58:02 and C⁄07:18) and facilitated the discovery of two novel HLA-A alleles, with intronic substitutions. For the remaining alleles, consensus sequences were compared against the reference sequences of the genotyped alleles and occasional differences between consensus sequences and reference sequences were manually inspected to evaluate algorithm performance. It has been determined that depth of coverage is critical in phasing and base calling, which is necessary to generate credible consensus sequences. Conclusions: Generation of high confidence HLA class I consensus sequences is possible using the Omixon Holotype HLA typing system provided that adequate depth of uniform coverage is obtained from sequencing. D. Monos: Other (Identify); Company/Organization; Omixon Royalties.