Rapid high-throughput human leukocyte antigen typing by massively parallel pyrosequencing for high-resolution allele identification

Gabriel, Christian; Danzer, Martin; Hackl, Christa; Kopal, Guido; Hufnagl, Peter; Hofer, Katja; Polin, Helene; Stabentheiner, Stephanie; Pröll, Johannes

doi:10.1016/j.humimm.2009.08.009

Cited by 87 publications

(60 citation statements)

References 26 publications

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“…Recently, several laboratories (16)(17)(18)(19)(20) have developed highthroughput HLA genotyping methodologies using massively parallel sequencing strategies such as Roche/454 sequencing (21). In all these high-throughput HLA-genotyping studies, with the exception of the study by Lind et al (19), a few polymorphic exons were amplified separately and sequenced in a multiplexed manner.…”

Section: Discussionmentioning

confidence: 99%

High-throughput, high-fidelity HLA genotyping with deep sequencing

Wang

Krishnakumar

Wilhelmy

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

177

144

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Human leukocyte antigen (HLA) genes are the most polymorphic in the human genome. They play a pivotal role in the immune response and have been implicated in numerous human pathologies, especially autoimmunity and infectious diseases. Despite their importance, however, they are rarely characterized comprehensively because of the prohibitive cost of standard technologies and the technical challenges of accurately discriminating between these highly related genes and their many allelles. Here we demonstrate a high-resolution, and cost-effective methodology to type HLA genes by sequencing, which combines the advantage of long-range amplification, the power of high-throughput sequencing platforms, and a unique genotyping algorithm. We calibrated our method for HLA-A, -B, -C, and -DRB1 genes with both reference cell lines and clinical samples and identified several previously undescribed alleles with mismatches, insertions, and deletions. We have further demonstrated the utility of this method in a clinical setting by typing five clinical samples in an Illumina MiSeq instrument with a 5-d turnaround. Overall, this technology has the capacity to deliver low-cost, high-throughput, and accurate HLA typing by multiplexing thousands of samples in a single sequencing run, which will enable comprehensive disease-association studies with large cohorts. Furthermore, this approach can also be extended to include other polymorphic genes.hematopoietic stem cell transplantation | sequence-based typing H uman leukocyte antigen (HLA) genes encode cell-surface proteins that bind and display fragments of antigens to T lymphocytes. This helps to initiate the adaptive immune response in higher vertebrates and thus is critical to the detection and identification of invading microorganisms (1). Six of the HLA genes (HLA-A, -B, -C, -DQA1, -DQB1, and -DRB1) are extremely polymorphic and constitute the most important set of markers for matching patients and donors for bone marrow transplantation (2, 3). Specific HLA alleles have been found to be associated with a number of autoimmune diseases, such as multiple sclerosis (4), narcolepsy (5), celiac disease (6), rheumatoid arthritis (7), and type I diabetes (3,8). Alleles have also been noted to be protective in infectious diseases such as HIV (9, 10), and numerous animal studies have shown that these genes are often the major contributors to disease susceptibility or resistance (11-13).HLA genes are among the most polymorphic in the human genome, and the changes in sequence affect the specificity of antigen presentation and histocompatibility in transplantation. A variety of methodologies have been developed for HLA typing at the protein and nucleic acid level. Whereas earlier HLA typing methods distinguished HLA antigens, modern methods such as sequence-based typing (SBT) determine the nucleotide sequences of HLA genes for higher resolution. However, due to cost and time constraints, HLA sequencing technologies have traditionally focused on the most polymorphic regions encoding the peptide-...

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Section: Discussionmentioning

confidence: 99%

High-throughput, high-fidelity HLA genotyping with deep sequencing

Wang

Krishnakumar

Wilhelmy

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

177

144

View full text Add to dashboard Cite

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“…• Multi-gene diagnostic panels (Morgan et al 2010) • Achieving a molecular diagnosis for rare genetic diseases (Lupski et al 2010;Ng et al 2010a, b;Worthey et al 2011;Vissers et al 2010) • Tissue matching and HLA-typing (Bentley et al 2009;Gabriel et al 2009;Lind et al 2010) • Non-invasive prenatal diagnosis (Chiu et al 2008;Fan et al 2008;Lo et al 2010) • Quantifying the burden of disease from solid tumours (Leary et al 2010;McBride et al 2010) and • Cancer genome profiling leading to stratified treatment regimens Diamandis et al 2010;Stratton et al 2009) RNA sequencing (RNA-seq) and chromatin immunoprecipitation (ChIP) sequencing can also be used to study gene expression and for detection of somatic mutations, gene fusions, and other non-mutational events, an understanding of which can have an impact on management of diseases such as cancer (Cowin et al 2010;Robison 2010). However, numerous barriers to clinical translation still exist, including: validation of the technology; standardisation of the analysis pipeline; integration of information from the numerous databases of genomic variation; building a robust evidence base to allow clinical interpretation of novel variants; developing a service delivery infrastructure that can capitalise upon the high-throughput advantages of new sequencing technologies; providing an appropriately skilled health care workforce to deal with genomic medicine; and addressing the numerous ethical, legal and social implications of sequencing, storing and accessing whole genomes.…”

Section: Resultsmentioning

confidence: 99%

Review of massively parallel DNA sequencing technologies

Moorthie

Mattocks

Wright

2011

HUGO J

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Since the development of technologies that can determine the base-pair sequence of DNA, the ability to sequence genes has contributed much to science and medicine. However, it has remained a relatively costly and laborious process, hindering its use as a routine biomedical tool. Recent times are seeing rapid developments in this field, both in the availability of novel sequencing platforms, as well as supporting technologies involved in processes such as targeting and data analysis. This is leading to significant reductions in the cost of sequencing a human genome and the potential for its use as a routine biomedical tool. This review is a snapshot of this rapidly moving field examining the current state of the art, forthcoming developments and some of the issues still to be resolved prior to the use of new sequencing technologies in routine clinical diagnosis.

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“…Sequence reads from related HLA genomic sequences that might be co-amplified along with the target sequence are automatically filtered out by the software, preventing ‘background' signals as it would occur in SBT or SSOP. This and further studies have shown that the amplicon sequencing strategy on the Roche 454 system is feasible for routine HLA typing, allows the resolution of cis-trans ambiguities, and facilitates a reliable determination of HLA genotypes at allelic level [15,30,31]. …”

Section: Hla Typing By Ngsmentioning

confidence: 99%

“…Gabriel et al [31] sequenced exons 2, 3, 4, of HLA-A and -B from 8 donor samples in one GS FLX run, resulting in an average coverage of 5,000 reads per amplicon for one sample. A significant number (44%) of the initial reads yet did not pass the internal quality control filters of the GS FLX analysis software, mainly due to short read lengths and mixed reads, as a result of the amplification of more than one specific DNA molecule on one bead in the emPCR.…”

Section: Hla Typing By Ngsmentioning

confidence: 99%

Diagnostic Applications of Next Generation Sequencing in Immunogenetics and Molecular Oncology

Grumbt¹,

Eck²,

Hinrichsen³

et al. 2013

Transfus Med Hemother

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With the introduction of the next generation sequencing (NGS) technologies, remarkable new diagnostic applications have been established in daily routine. Implementation of NGS is challenging in clinical diagnostics, but definite advantages and new diagnostic possibilities make the switch to the technology inevitable. In addition to the higher sequencing capacity, clonal sequencing of single molecules, multiplexing of samples, higher diagnostic sensitivity, workflow miniaturization, and cost benefits are some of the valuable features of the technology. After the recent advances, NGS emerged as a proven alternative for classical Sanger sequencing in the typing of human leukocyte antigens (HLA). By virtue of the clonal amplification of single DNA molecules ambiguous typing results can be avoided. Simultaneously, a higher sample throughput can be achieved by tagging of DNA molecules with multiplex identifiers and pooling of PCR products before sequencing. In our experience, up to 380 samples can be typed for HLA-A, -B, and -DRB1 in high-resolution during every sequencing run. In molecular oncology, NGS shows a markedly increased sensitivity in comparison to the conventional Sanger sequencing and is developing to the standard diagnostic tool in detection of somatic mutations in cancer cells with great impact on personalized treatment of patients.

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Rapid high-throughput human leukocyte antigen typing by massively parallel pyrosequencing for high-resolution allele identification

Cited by 87 publications

References 26 publications

High-throughput, high-fidelity HLA genotyping with deep sequencing

High-throughput, high-fidelity HLA genotyping with deep sequencing

Review of massively parallel DNA sequencing technologies

Diagnostic Applications of Next Generation Sequencing in Immunogenetics and Molecular Oncology

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