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BACKGROUND: Next-generation sequencing (NGS) has changed genomics significantly. More and more applications strive for sequencing with different platforms. Now, in 2012, after a decade of development and evolution, NGS has been accepted for a variety of research fields. Determination of sequencing errors is essential in order to follow next-generation sequencing beyond research use only. This study describes the overall 454 system performance of using multiple GS Junior runs with an in-house established and validated diagnostic assay for human leukocyte antigen (HLA) exon sequencing. Based on this data, we extracted, evaluated and characterized errors and variants of 60 HLA loci per run with respect to their adjacencies. RESULTS: We determined an overall error rate of 0.18% in a total of 118,484,408 bases. 31.3% of all reads analyzed (n=349,503) contain one or more errors. The largest group are deletions that account for 50% of the errors. Incorrect bases are not distributed equally along sequences and tend to be more frequent at sequence ends. Certain sequence positions in the middle or at the beginning of the read accumulate errors. Typically, the corresponding quality score at the actual error position is lower than the adjacent scores. CONCLUSIONS: Here we present the first error assessment in a human next-generation sequencing diagnostics assay in an amplicon sequencing approach. Improvements of sequence quality and error rate that have been made over the years are evident and it is shown that both have now reached a level where diagnostic applications become feasible. Our presented data are better than previously published error rates and we can confirm and quantify the often described relation of homopolymers and errors. Nevertheless, a certain depth of coverage is needed, in particular with challenging areas of the sequencing target. Furthermore, the usage of error correcting tools is not essential but might contribute towards the capacity and efficiency of a sequencing run.
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Exones , Antígenos HLA/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Prueba de Histocompatibilidad , Análisis de Secuencia de ADN/métodos , HumanosRESUMEN
BACKGROUND: Human leukocyte antigen matching at allelic resolution is proven clinically significant in hematopoietic stem cell transplantation, lowering the risk of graft-versus-host disease and mortality. However, due to the ever growing HLA allele database, tissue typing laboratories face substantial challenges. In light of the complexity and the high degree of allelic diversity, it has become increasingly difficult to define the classical transplantation antigens at high-resolution by using well-tried methods. Thus, next-generation sequencing is entering into diagnostic laboratories at the perfect time and serving as a promising tool to overcome intrinsic HLA typing problems. Therefore, we have developed and validated a scalable automated HLA class I and class II typing approach suitable for diagnostic use. RESULTS: A validation panel of 173 clinical and proficiency testing samples was analysed, demonstrating 100% concordance to the reference method. From a total of 1,273 loci we were able to generate 1,241 (97.3%) initial successful typings. The mean ambiguity reduction for the analysed loci was 93.5%. Allele assignment including intronic sequences showed an improved resolution (99.2%) of non-expressed HLA alleles. CONCLUSION: We provide a powerful HLA typing protocol offering a short turnaround time of only two days, a fully integrated workflow and most importantly a high degree of typing reliability. The presented automated assay is flexible and can be scaled by specific primer compilations and the use of different 454 sequencing systems. The workflow was successfully validated according to the policies of the European Federation for Immunogenetics. Next-generation sequencing seems to become one of the new methods in the field of Histocompatibility.
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Antígenos HLA/análisis , Prueba de Histocompatibilidad/métodos , Automatización de Laboratorios/métodos , Humanos , Tipificación de Secuencias Multilocus/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Reproducibilidad de los ResultadosRESUMEN
Genotypic HIV-1 drug resistance testing with standard Sanger sequencing is limited to the detection of mutations with >20% prevalence. A new protocol for variant detection of protease and reverse transcriptase genes of HIV-1 genotype B samples with ultra-deep sequencing on the GS-FLX sequencer (Roche 454 Life Sciences, Branford, CT) was evaluated. The new technology was compared with the standard Sanger sequencing method. For accuracy testing, genotype B samples obtained from proficiency panels were examined with ultra-deep sequencing. Reproducibility was determined by repeat GS-FLX sequencing of 21 clinical samples. Clinical performance was evaluated with 44 samples and the results were compared to the TRUGENE HIV-1 Genotyping Kit (Siemens Healthcare Diagnostics, Tarrytown, NY). Sequences generated with both protocols were analyzed using the Stanford University HIV drug resistance database. When accuracy was tested, 316 of 317 mutation codons included in the analysis of proficiency panels could be identified correctly with ultra-deep sequencing. Reproducibility testing resulted in a correlation value of R(2)=0.969. Analysis of 44 routine clinical samples with the Stanford University HIV drug resistance database revealed a total number of 269 and 171 mutations by the ultra-deep and standard Sanger sequencing, respectively. Drug resistance interpretations showed differences for 11 samples. With ultra-deep sequencing, total time to result was four times longer in comparison to standard Sanger sequencing. Manual work was increased significantly using the new protocol. The ultra-deep sequencing protocol showed good accuracy and reproducibility. However, automation and shorter time to obtain results are essential for use in the routine diagnostic laboratory.
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Fármacos Anti-VIH/farmacología , Farmacorresistencia Viral , VIH-1/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Automatización/métodos , Genotipo , Proteasa del VIH/genética , Transcriptasa Inversa del VIH/genética , VIH-1/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana/métodos , Mutación Missense , Reproducibilidad de los Resultados , Factores de TiempoRESUMEN
How cells coordinate the immune system activities is important for potentially life-saving organ or stem cell transplantations. Polymorphic immunoregulatory genes, many of them located in the human major histocompatibility complex, impact the process and assure the proper execution of tolerance-versus-activity mechanisms. In haematopoietic stem cell transplantation, on the basis of fully human leukocyte antigen (HLA)-matched donor-recipient pairs, adverse effects like graft versus leukaemia and graft versus host are observed and difficult to handle. So far, high-resolution HLA typing was performed with Sanger sequencing, but for methodological reasons information on additional immunocompetent major histocompatibility complex loci has not been revealed. Now, we have used microarray sequence capture and targeted enrichment combined with next generation pyrosequencing for 3.5 million base pair human major histocompatibility complex resequencing in a clinical transplant setting and describe 3025 variant single nucleotide polymorphisms, insertions and deletions among recipient and donor in a single sequencing experiment. Taken together, the presented data show that sequence capture and massively parallel pyrosequencing can be used as a new tool for risk assessment in the setting of allogeneic stem cell transplantation.
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Epítopos/genética , Antígenos HLA/genética , Trasplante de Células Madre Hematopoyéticas , Secuenciación de Nucleótidos de Alto Rendimiento , Análisis de Secuencia de ADN , Enfermedad Injerto contra Huésped/genética , Prueba de Histocompatibilidad , Humanos , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
The human genome project triggered the introduction of next generation sequencing (NGS) systems. Although originally developed for total genome sequencing, metagenomics and plant genetics, the ultra-deep sequencing feature of NGS was utilized for diagnostic purposes in HIV resistance and tropism as well in detecting new mutations and tumor clones in oncology. Recent publications exploited the feature of clonal sequencing for immunogenetics to dissolve the growing number of ambiguities. This concept is quite reliable if all exons of interest are tested and the amplification region includes flanking introns. Challenging questions on quality control, cost effectiveness, workflow, and management of enormous loads of data remain if NGS is considered as routine method in the immunogenetics laboratory. If solved, NGS has big potential to have a major impact on immunogenetics by way of providing ambiguity-free HLA-typing results faster, but will also have a great influence on how immunogenetics testing and workflows are organized.
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Transplantation and, notably, hematopoietic stem cell transplantation require high-resolution human leukocyte antigen (HLA) typing and, because of the heterozygous genomic DNA samples, are dependent on clonal analytical methods. High-resolution HLA typing is a necessity for accomplishing the best possible histocompatibility match between donor and recipient, because mismatches strongly increase the risk of severe acute graft-versus-host disease. We describe the development and first application in a clinical setting of a novel, HLA sequence-based typing method by exploring the next-generation sequencing technology as provided by the Genome Sequencer FLX system (Roche/454 Life Sciences, Branford, CT). The developed system allows for ambiguity-free, high-throughput, high-resolution HLA-A and -B typing with the potential for automation. Primers and Genome Sequencer FLX specific adapters were lengthened with donor-identifying barcode sequences to identify each of eight Caucasian reference donors within one single multiplex sequencing run. Compared with normal SBT HLA typing, results indicate that every patient was identified correctly with an average of 1000 reads per amplicon. Furthermore, current investments for increased read lengths and fully automated molecular diagnostic software tools, using original GS-FLX data file formats, will enhance this novel HLA typing strategy in the near future.
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Alelos , Antígenos HLA/análisis , Antígenos HLA/genética , Ensayos Analíticos de Alto Rendimiento/métodos , Prueba de Histocompatibilidad/métodos , Secuencia de Bases , Genoma Humano , Humanos , Datos de Secuencia Molecular , Factores de TiempoRESUMEN
BACKGROUND: Hematopoietic stem-cell transplantation is a well-established treatment in various hematologic malignancies, but the outcome depends on disease relapse, infections, and the development and severity of acute and chronic graft-versus-host disease. Some evidence has revealed an important role for the nonclassical major histocompatibility complex class I molecules in transplantation, most notably human leukocyte antigen (HLA)-E. This study evaluates the impact of HLA-E alleles on transplantation outcome after HLA-matched allogeneic HSCT. METHODS: We genotyped DNA for HLA-E polymorphism from 83 recipients and their respective donors by real-time polymerase chain reaction after melting curve analysis and compared the results with clinical outcome. RESULTS: HLA-E*0103 homozygous patients showed a higher probability of overall survival (P=0.003) and disease-free survival (P=0.001) in a univariate model. Cox regression analysis confirmed HLA-E*0103, 0103 (P=0.006; relative risk 1.12; 95% confidence interval 0.31-1.94) and early stage of disease (P=0.005; relative risk 1.16; 95% confidence interval 0.45-1.86) as independent factors improving overall survival. Moreover, homozygosity for HLA-E*0103 was associated with a significant decreased incidence of transplant-related mortality (P=0.01). CONCLUSIONS: We found an association between HLA-E*0103 homozygosity and the significant reduction of transplant-related mortality in related and unrelated HSCT. The risk of posttransplant complications was significantly reduced when the donor possesses the HLA-E*0103, 0103 genotype, and this was translated in a better overall survival.
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Antígenos HLA/genética , Trasplante de Células Madre Hematopoyéticas , Enfermedad Aguda , Adulto , Anciano , Secuencia de Bases , Estudios de Cohortes , Cartilla de ADN/genética , Femenino , Genotipo , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/inmunología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Trasplante de Células Madre Hematopoyéticas/mortalidad , Heterocigoto , Antígenos de Histocompatibilidad Clase I , Homocigoto , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Trasplante Homólogo , Adulto Joven , Antígenos HLA-ERESUMEN
The accumulation of chromosomal aberrations is a characteristic feature of tumor development. However, an understanding of tumorigenesis that assumes that changes in DNA copy number always cause equivalent changes in the corresponding RNA and protein levels is an oversimplification and completely ignores the individual genetic and epigenetic context in which an aberration has to be evaluated. We present a brief introduction to various techniques dedicated to the genome-wide analysis of genetic and epigenetic changes, and illustrate how complementary information derived from these various DNA array-based technologies can lead to a better understanding of the consequences of chromosomal aberrations.