Objective:To analyze and confirm the ambiguous results of HLA-DRB1 genotyping in one case.Methods:HLA genotyping was performed on a sample of hematopoietic stem cell donor using Illumina MiSeq-based next-generation sequencing(NGS).The ambiguous results of HLA-DRB1 locus were further analyzed and confirmed through PacBio SMRT third-generation sequencing.Results:The Illumina MiSeq-based NGS typing results suggested the presence of a new HLA-DRB1*11 allele(DRB1*11:NEW,12:01)in the specimen,with a mismatch of G＞A located in the 40th residue of exon 1 compared with the nearest allele DRB1*11:01:01:03.However,due to the long sequence of intron 1,this observed mutation site was so far away from the near heterozygous sites that no reads could cover this gap.Therefore,it was impossible to determine which consensus the mutation site was located in,and the NGS-based genotyping results were obtained from the random allocation by the software,which was ambiguous and unreliable.In order to confirm the results,the long-read third generation sequencing technology based on PacBio was applied to genotype the DRB1 locus.The results showed that the DRB1 typing was HLA-DRB1*11:01,12:10.E1-40A was actually located in the allele HLA-DRB1*12:XX,which was exactly matched with HLA-DRB1*12:10.Conclusion:For some new alleles suggested by NGS,especially the ambiguous ones that are far away from other heterozygous sites,it is necessary to analyze and confirm them by other methods such as the third-generation long-read sequencing technology to obtain reliable results.

Objective:To analyze and confirm the ambiguous results of HLA-DRB1 genotyping in one case.Methods:HLA genotyping was performed on a sample of hematopoietic stem cell donor using Illumina MiSeq-based next-generation sequencing(NGS).The ambiguous results of HLA-DRB1 locus were further analyzed and confirmed through PacBio SMRT third-generation sequencing.Results:The Illumina MiSeq-based NGS typing results suggested the presence of a new HLA-DRB1*11 allele(DRB1*11:NEW,12:01)in the specimen,with a mismatch of G＞A located in the 40th residue of exon 1 compared with the nearest allele DRB1*11:01:01:03.However,due to the long sequence of intron 1,this observed mutation site was so far away from the near heterozygous sites that no reads could cover this gap.Therefore,it was impossible to determine which consensus the mutation site was located in,and the NGS-based genotyping results were obtained from the random allocation by the software,which was ambiguous and unreliable.In order to confirm the results,the long-read third generation sequencing technology based on PacBio was applied to genotype the DRB1 locus.The results showed that the DRB1 typing was HLA-DRB1*11:01,12:10.E1-40A was actually located in the allele HLA-DRB1*12:XX,which was exactly matched with HLA-DRB1*12:10.Conclusion:For some new alleles suggested by NGS,especially the ambiguous ones that are far away from other heterozygous sites,it is necessary to analyze and confirm them by other methods such as the third-generation long-read sequencing technology to obtain reliable results.

Objective:To confirm the sequence of a null allele HLA-C*08:127N produced by a base insertion.Methods:PCR sequence-specific oligonucleotide probe(SSOP)and PCR sequence-based typing(SBT)were used for HLA routine detection,which discovered abnormal sequence maps of HLA-C in one acute myeloid leukemia patient.The sequence of the above loci was confirmed by next generation sequencing(NGS)technology.Results:The SSOP typing result showed that HLA-C locus was C*03:04,C*08:01,while the sequence was suspected to be inserted or deleted in exon 3 by SBT,and finally confirmed by NGS as C*03:04,C*08:127N.Conclusion:When base insertion produces HLA null alleles,SBT analysis software cannot provide correct results,but NGS technology can more intuitively obtain accurate HLA typing results.

Objective:To investigate the accuracy of next-generation sequencing technology(NGS)in detecting the polymorphisms of HLA-DRB1,DQB1,DQA1,DRB3,DRB4,DRB5,DPA1 and DPB1 alleles in randomly-selected unrelated healthy individuals from Shenzhen Han population,investigate the potential reason for HLA-DRB1 allele dropout in routine NGS,and establish an internal quality control system.Methods:NGS-based HLA class Ⅱ genotyping was performed on 1 012 samples using the MiSeqDxTM platform.The suspected missed alleles indicated by the quality control software and HLA-DRB1 homozygotes were confirmed by PCR-SSOP or PCR-SBT methods.Results:A total of 139 alleles were detected,including HLA-DRB1(45),DRB3(7),DRB4(5),DRB5(7),DQA1(17),DQB1(21),DPA1(10)and DPB1(27).HLA-DRB 1*09:01(17.09％),15:01(10.72％);DRB3*02:02(25.99％),03:01(10.18％);DRB4*01:03(36.46％);DRB5*01:01(15.42％);DQA1*01:02(20.01％),03:02(17.19％);DQB1*03:01(19.47％),03:03(17.98％),05:02(11.66％),06:01(10.67％);DPA1*02:02(54.45％),01:03(31.18％)and DPB1*05:01(39.13％),02:01(16.90％)alleles were the most common alleles in Shenzhen Han population(frequencies＞10％).There was no statistical difference between the gene frequencies of HLA-DRB1 and DQB1 loci in our study.The HLA Common and Well-Documented Alleles in China(CWD2.4)(x2=12.68,P＞0.05).94 cases of HLA-DRB1 homozygous samples detected by NGS were retested by PCR-SSOP or SBT method,and one case of allele dropout at HLA-DRB1 locus was found.SBT method confirmed that the allele of DRB1*04:03 was missed.The laboratory internal quality control system was established.Two cases of new alleles were detected and named by WHO Nomenclature Committee for Factors of the HLA System.Conclusion:The HLA genotyping results based on NGS showed a significantly lower ambiguity rate.The HLA class Ⅱ alleles exhibit genetic polymorphism in the Han population of unrelated healthy individuals in Shenzhen.The independent method based on NGS in clinical histocompatibility testing has limitations and requires internal quality control strategies to avoid allele-dropout events.

OBJECTIVE: To confirm the HLA genotypes of the samples including 4 cases of magnetic bead probe HLA genotyping result pattern abnormality and 3 cases of ambiguous result detected by PCR sequence-specific oligonudeotide probe (SSOP) method. METHODS: All samples derived from HLA high-resolution typing laboratory were detected by PCR-SSOP. A total of 4 samples of magnetic bead probe HLA genotyping result pattern abnormality and 3 samples of ambiguous result were further confirmed by PCR sequence-based typing (SBT) technology and next-generation sequencing (NGS) technology. RESULTS: A total of 4 samples of magnetic bead probe HLA genotyping result pattern abnormality were detected by PCR-SSOP method. The results of SBT and NGS showed that the HLA-A genotype of sample 1 did not match any known genotypes. NGS analysis revealed that the novel allele was different from the closest matching allele A*31:01:02:01at position 154 with G>A in exon 2, which resulting in one amino acid substitution at codon 28 from Valine to Methionine (p.Val28Met). The HLA-C genotype of sample 2 was C*03:119, 06:02, sample 3 was C*03:03, 07:137, and sample 4 was B*55:02, 55:12. A total of 3 samples with ambiguous result were initially detected by PCR-SSOP method. The re-examination results of SBT and NGS showed that the HLA-B genotype of sample 5 was B*15:58, 38:02, sample 6 was DRB1*04:05, 14:101, and sample 7 was DQB1*03:34, 05:02. Among them, alleles C*03:119, C*07:137 and DRB1*14:101 were not included in the Common and Well-documented Alleles (CWD) v2.4 of the Chinese Hematopoietic Stem Cell Donor Database. CONCLUSION The abnormal pattern of HLA genotyping results of magnetic probe by PCR-SSOP method suggests that it may be a rare allele or a novel allele, which needs to be verified by sequencing.
Humans ; Alleles ; Polymerase Chain Reaction ; Genotype ; High-Throughput Nucleotide Sequencing ; Histocompatibility Testing/methods* ; Technology