Design and Validation of a Multiplex KIR and HLA Class I Genotyping Method Using Next Generation Sequencing
Killer cell immunoglobulin-like receptors (KIR), considered the most polymorphic natural killer (NK) cell regulators, bind HLA class-I molecules or still unknown ligands. Interest in KIR genotyping is increasing because of the importance of these receptors for identifying the best possible donor in...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2018-12-01
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| Series: | Frontiers in Immunology |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fimmu.2018.02991/full |
| _version_ | 1818901140957298688 |
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| author | Laia Closa Laia Closa Francisco Vidal Francisco Vidal Francisco Vidal Maria J. Herrero Jose L. Caro Jose L. Caro |
| author_facet | Laia Closa Laia Closa Francisco Vidal Francisco Vidal Francisco Vidal Maria J. Herrero Jose L. Caro Jose L. Caro |
| author_sort | Laia Closa |
| collection | DOAJ |
| description | Killer cell immunoglobulin-like receptors (KIR), considered the most polymorphic natural killer (NK) cell regulators, bind HLA class-I molecules or still unknown ligands. Interest in KIR genotyping is increasing because of the importance of these receptors for identifying the best possible donor in hematopoietic stem cell transplantation to obtain a graft-versus-leukemia effect. Currently, routine protocols to determine the gene content of the KIR cluster are exclusively performed by PCR-SSO and PCR-SSP. To improve the study of these genes, we developed a multiplex, long-range PCR strategy suitable for simultaneous, high-resolution HLA class I and KIR genotyping by next generation sequencing (NGS). This protocol allows amplification of the 14 KIR genes, 2 KIR pseudogenes, and HLA class I genes, with subsequent sequencing on an Illumina platform. The bioinformatics analysis for KIR genotyping was performed by virtual hybridization of gene-specific probes, and HLA genotyping was done by GenDx NGSengine software. To validate the method reliability, 192 genomic DNA samples previously characterized by PCR-SSO were used. When a specific KIR gene was present, a large number of gene-specific virtual probes were detected, whereas when it was absent, very few or none were found, enabling cutoff establishment. Concordance for both the KIR and HLA assignments as compared with the previous characterization was 100%. In conclusion, the multiplex PCR NGS-based strategy presented could provide an efficient, less costly method for KIR-ligand genotyping by gene presence/absence. Furthermore, allele resolution will be possible when KIR-specific software becomes available. |
| first_indexed | 2024-12-19T20:15:02Z |
| format | Article |
| id | doaj.art-cc70104b30fa41b4a38aa7932bd8941f |
| institution | Directory Open Access Journal |
| issn | 1664-3224 |
| language | English |
| last_indexed | 2024-12-19T20:15:02Z |
| publishDate | 2018-12-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Immunology |
| spelling | doaj.art-cc70104b30fa41b4a38aa7932bd8941f2022-12-21T20:07:12ZengFrontiers Media S.A.Frontiers in Immunology1664-32242018-12-01910.3389/fimmu.2018.02991422246Design and Validation of a Multiplex KIR and HLA Class I Genotyping Method Using Next Generation SequencingLaia Closa0Laia Closa1Francisco Vidal2Francisco Vidal3Francisco Vidal4Maria J. Herrero5Jose L. Caro6Jose L. Caro7Histocompatibility and Immunogenetics Laboratory, Blood and Tissue Bank, Barcelona, SpainTransfusional Medicine Group, Vall d'Hebron Research Institute, Barcelona, SpainTransfusional Medicine Group, Vall d'Hebron Research Institute, Barcelona, SpainCongenital Coagulopathy Laboratory, Blood and Tissue Bank, Barcelona, SpainCIBER of Cardiovascular Diseases, Madrid, SpainHistocompatibility and Immunogenetics Laboratory, Blood and Tissue Bank, Barcelona, SpainHistocompatibility and Immunogenetics Laboratory, Blood and Tissue Bank, Barcelona, SpainTransfusional Medicine Group, Vall d'Hebron Research Institute, Barcelona, SpainKiller cell immunoglobulin-like receptors (KIR), considered the most polymorphic natural killer (NK) cell regulators, bind HLA class-I molecules or still unknown ligands. Interest in KIR genotyping is increasing because of the importance of these receptors for identifying the best possible donor in hematopoietic stem cell transplantation to obtain a graft-versus-leukemia effect. Currently, routine protocols to determine the gene content of the KIR cluster are exclusively performed by PCR-SSO and PCR-SSP. To improve the study of these genes, we developed a multiplex, long-range PCR strategy suitable for simultaneous, high-resolution HLA class I and KIR genotyping by next generation sequencing (NGS). This protocol allows amplification of the 14 KIR genes, 2 KIR pseudogenes, and HLA class I genes, with subsequent sequencing on an Illumina platform. The bioinformatics analysis for KIR genotyping was performed by virtual hybridization of gene-specific probes, and HLA genotyping was done by GenDx NGSengine software. To validate the method reliability, 192 genomic DNA samples previously characterized by PCR-SSO were used. When a specific KIR gene was present, a large number of gene-specific virtual probes were detected, whereas when it was absent, very few or none were found, enabling cutoff establishment. Concordance for both the KIR and HLA assignments as compared with the previous characterization was 100%. In conclusion, the multiplex PCR NGS-based strategy presented could provide an efficient, less costly method for KIR-ligand genotyping by gene presence/absence. Furthermore, allele resolution will be possible when KIR-specific software becomes available.https://www.frontiersin.org/article/10.3389/fimmu.2018.02991/fullKIRHLAnext generation sequencinggenotypingNGS analysis |
| spellingShingle | Laia Closa Laia Closa Francisco Vidal Francisco Vidal Francisco Vidal Maria J. Herrero Jose L. Caro Jose L. Caro Design and Validation of a Multiplex KIR and HLA Class I Genotyping Method Using Next Generation Sequencing Frontiers in Immunology KIR HLA next generation sequencing genotyping NGS analysis |
| title | Design and Validation of a Multiplex KIR and HLA Class I Genotyping Method Using Next Generation Sequencing |
| title_full | Design and Validation of a Multiplex KIR and HLA Class I Genotyping Method Using Next Generation Sequencing |
| title_fullStr | Design and Validation of a Multiplex KIR and HLA Class I Genotyping Method Using Next Generation Sequencing |
| title_full_unstemmed | Design and Validation of a Multiplex KIR and HLA Class I Genotyping Method Using Next Generation Sequencing |
| title_short | Design and Validation of a Multiplex KIR and HLA Class I Genotyping Method Using Next Generation Sequencing |
| title_sort | design and validation of a multiplex kir and hla class i genotyping method using next generation sequencing |
| topic | KIR HLA next generation sequencing genotyping NGS analysis |
| url | https://www.frontiersin.org/article/10.3389/fimmu.2018.02991/full |
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