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Adams, Sharon; Barracchini, Kathleen C.; Chen, Deborah; Robbins, Fu-Meei; Wang, Lu; Larsen, Paula; Luhm, Robert A.; Stroncek, David F.

doi:10.1186/1479-5876-2-30

Cited by 64 publications

(17 citation statements)

References 7 publications

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“…Conventional PCR-based genotyping approaches incorporating restriction fragment length polymorphism (PCR-RFLP) ( 9 ), single strand conformation polymorphism (PCR-SSCP) ( 10 ), sequence-specific oligonucleotides (PCR-SSO) ( 11 ), sequence-specific primers (PCR-SSP) ( 12 ), and Sanger sequencing-based typing (PCR-SBT) ( 13 ) have been used for HLA-testing in disease association and pre-transplantation ( 14 – 16 ) analysis. However, these methods are limited in their ability to decipher chromosomal phase ( cis/trans ) ambiguity and/or imprecise allele identification ( 17 , 18 ) and may leave multiple pairs of HLA gene alleles unresolved. Moreover, the traditional methods focus only on the variations in the highly polymorphic regions of the HLA genes, and thus the majority of the assays only interrogate exons 2 and 3 or exons 2, 3, and 4 of the class I loci and exons 2 or exons 2 and 3 of class II.…”

Section: Introductionmentioning

confidence: 99%

Reference Grade Characterization of Polymorphisms in Full-Length HLA Class I and II Genes With Short-Read Sequencing on the ION PGM System and Long-Reads Generated by Single Molecule, Real-Time Sequencing on the PacBio Platform

Suzuki

Ranade

Osaki³

et al. 2018

Front. Immunol.

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Although NGS technologies fuel advances in high-throughput HLA genotyping methods for identification and classification of HLA genes to assist with precision medicine efforts in disease and transplantation, the efficiency of these methods are impeded by the absence of adequately-characterized high-frequency HLA allele reference sequence databases for the highly polymorphic HLA gene system. Here, we report on producing a comprehensive collection of full-length HLA allele sequences for eight classical HLA loci found in the Japanese population. We augmented the second-generation short read data generated by the Ion Torrent technology with long amplicon spanning consensus reads delivered by the third-generation SMRT sequencing method to create reference grade high-quality sequences of HLA class I and II gene alleles resolved at the genomic coding and non-coding level. Forty-six DNAs were obtained from a reference set used previously to establish the HLA allele frequency data in Japanese subjects. The samples included alleles with a collective allele frequency in the Japanese population of more than 99.2%. The HLA loci were independently amplified by long-range PCR using previously designed HLA-locus specific primers and subsequently sequenced using SMRT and Ion PGM sequencers. The mapped long and short-reads were used to produce a reference library of consensus HLA allelic sequences with the help of the reference-aware software tool LAA for SMRT Sequencing. A total of 253 distinct alleles were determined for 46 healthy subjects. Of them, 137 were novel alleles: 101 SNVs and/or indels and 36 extended alleles at a partial or full-length level. Comparing the HLA sequences from the perspective of nucleotide diversity revealed that HLA-DRB1 was the most divergent among the eight HLA genes, and that the HLA-DPB1 gene sequences diverged into two distinct groups, DP2 and DP5, with evidence of independent polymorphisms generated in exon 2. We also identified two specific intronic variations in HLA-DRB1 that might be involved in rheumatoid arthritis. In conclusion, full-length HLA allele sequencing by third-generation and second-generation technologies has provided polymorphic gene reference sequences at a genomic allelic resolution including allelic variations assigned up to the field-4 level for a stronger foundation in precision medicine and HLA-related disease and transplantation studies.

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Section: Introductionmentioning

confidence: 99%

Reference Grade Characterization of Polymorphisms in Full-Length HLA Class I and II Genes With Short-Read Sequencing on the ION PGM System and Long-Reads Generated by Single Molecule, Real-Time Sequencing on the PacBio Platform

Suzuki

Ranade

Osaki³

et al. 2018

Front. Immunol.

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“…The HLA genotyping methods mainly applied today are PCR-SSOP, such as the Luminex commercial methodology [ 25 , 26 ], and SBT by the Sanger method employing capillary sequencing based on chain-termination reactions [ 14 , 27 ]. However, both methods often detect more than one pair of unresolved HLA alleles because of chromosomal phase ( cis/trans ) ambiguity [ 28 - 30 ]. To solve the phase ambiguity problem, we previously reported the development and application of the super high resolution-single molecule-sequence-based typing (SS-SBT) method using long-range PCR of the sample DNA from the promoter-enhancer region to the 3′ untranslated region (3′UTR) for 11 classical HLA loci, HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DRB3/4/5, HLA-DQA1, HLA-DQB1, HLA-DPA1, and HLA-DPB1 in combination with next generation sequencing (NGS) platforms such as Ion PGM (Life Technologies) and GS Junior (Roche) [ 31 - 33 ].…”

Section: Introductionmentioning

confidence: 99%

Cost-efficient multiplex PCR for routine genotyping of up to nine classical HLA loci in a single analytical run of multiple samples by next generation sequencing

et al. 2015

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BackgroundHLA genotyping by next generation sequencing (NGS) requires three basic steps, PCR, NGS, and allele assignment. Compared to the conventional methods, such as PCR-sequence specific oligonucleotide primers (SSOP) and -sequence based typing (SBT), PCR-NGS is extremely labor intensive and time consuming. In order to simplify and accelerate the NGS-based HLA genotyping method for multiple DNA samples, we developed and evaluated four multiplex PCR methods for genotyping up to nine classical HLA loci including HLA-A, HLA-B, HLA-C, HLA-DRB1/3/4/5, HLA-DQB1, and HLA-DPB1.ResultsWe developed multiplex PCR methods using newly and previously designed middle ranged PCR primer sets for genotyping different combinations of HLA loci, (1) HLA-DRB1/3/4/5, (2) HLA-DQB1 (3.8 kb to 5.3 kb), (3) HLA-A, HLA-B, HLA-C, and (4) HLA-DPB1 (4.6 kb to 7.2 kb). The primer sets were designed to genotype polymorphic exons to the field 3 level or 6-digit typing. When we evaluated the PCR method for genotyping all nine HLA loci (9LOCI) using 46 Japanese reference subjects who represented a distribution of more than 99.5% of the HLA alleles at each of the nine HLA loci, all of the 276 alleles genotyped, except for HLA-DRB3/4/5 alleles, were consistent with known alleles assigned by the conventional methods together with relevant locus balance and no excessive allelic imbalance. One multiplex PCR method (9LOCI) was able to provide precise genotyping data even when only 1 ng of genomic DNA was used for the PCR as a sample template.ConclusionsIn this study, we have demonstrated that the multiplex PCR approach for NGS-based HLA genotyping could serve as an alternative routine HLA genotyping method, possibly replacing the conventional methods by providing an accelerated yet robust amplification step. The method also could provide significant merits for clinical applications with its ability to amplify lower quantity of samples and the cost-saving factors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1514-4) contains supplementary material, which is available to authorized users.

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“…Breaking the cis -linkage among SNPs is one of the main drawbacks of some HLA typing methods (e.g., SBT) as it can hamper the typing of heterozygous individuals [43]. By basing the novel platform on the PCR-SSP method, we were able to preserve both the information about the polymorphisms and their linkage.…”

Section: Discussionmentioning

confidence: 99%

High-Throughput High-Resolution Class I HLA Genotyping in East Africa

et al. 2010

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HLA, the most genetically diverse loci in the human genome, play a crucial role in host-pathogen interaction by mediating innate and adaptive cellular immune responses. A vast number of infectious diseases affect East Africa, including HIV/AIDS, malaria, and tuberculosis, but the HLA genetic diversity in this region remains incompletely described. This is a major obstacle for the design and evaluation of preventive vaccines. Available HLA typing techniques, that provide the 4-digit level resolution needed to interpret immune responses, lack sufficient throughput for large immunoepidemiological studies. Here we present a novel HLA typing assay bridging the gap between high resolution and high throughput. The assay is based on real-time PCR using sequence-specific primers (SSP) and can genotype carriers of the 49 most common East African class I HLA-A, -B, and -C alleles, at the 4-digit level. Using a validation panel of 175 samples from Kampala, Uganda, previously defined by sequence-based typing, the new assay performed with 100% sensitivity and specificity. The assay was also implemented to define the HLA genetic complexity of a previously uncharacterized Tanzanian population, demonstrating its inclusion in the major East African genetic cluster. The availability of genotyping tools with this capacity will be extremely useful in the identification of correlates of immune protection and the evaluation of candidate vaccine efficacy.

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Cited by 64 publications

References 7 publications

Reference Grade Characterization of Polymorphisms in Full-Length HLA Class I and II Genes With Short-Read Sequencing on the ION PGM System and Long-Reads Generated by Single Molecule, Real-Time Sequencing on the PacBio Platform

Reference Grade Characterization of Polymorphisms in Full-Length HLA Class I and II Genes With Short-Read Sequencing on the ION PGM System and Long-Reads Generated by Single Molecule, Real-Time Sequencing on the PacBio Platform

Cost-efficient multiplex PCR for routine genotyping of up to nine classical HLA loci in a single analytical run of multiple samples by next generation sequencing

High-Throughput High-Resolution Class I HLA Genotyping in East Africa

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