RESUMEN
We evaluated multiplex PCR amplification as a front-end for high-throughput sequencing, to widen the applicability of massive parallel sequencers for the detailed analysis of complex genomes. Using multiplex PCR reactions, we sequenced the complete coding regions of seven genes implicated in peripheral neuropathies in 40 individuals on a GS-FLX genome sequencer (Roche). The resulting dataset showed highly specific and uniform amplification. Comparison of the GS-FLX sequencing data with the dataset generated by Sanger sequencing confirmed the detection of all variants present and proved the sensitivity of the method for mutation detection. In addition, we showed that we could exploit the multiplexed PCR amplicons to determine individual copy number variation (CNV), increasing the spectrum of detected variations to both genetic and genomic variants. We conclude that our straightforward procedure substantially expands the applicability of the massive parallel sequencers for sequencing projects of a moderate number of amplicons (50-500) with typical applications in resequencing exons in positional or functional candidate regions and molecular genetic diagnostics.
Asunto(s)
Dosificación de Gen , Mutación , Reacción en Cadena de la Polimerasa/métodos , Análisis de Secuencia de ADN/métodos , Enfermedad de Charcot-Marie-Tooth/diagnóstico , Enfermedad de Charcot-Marie-Tooth/genética , Conexinas/genética , Proteína 2 de la Respuesta de Crecimiento Precoz/genética , GTP Fosfohidrolasas , Predisposición Genética a la Enfermedad/genética , Variación Genética , Humanos , Proteínas de la Membrana/genética , Proteínas Mitocondriales/genética , Proteína P0 de la Mielina/genética , Proteínas de la Mielina/genética , Proteínas de Neurofilamentos/genética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Análisis de Secuencia de ADN/instrumentación , Proteína beta1 de Unión ComunicanteRESUMEN
OBJECTIVE: The multifunctional cytokine IL-13 is thought to play a central role in Type 2 inflammation in asthma. Serum periostin has been explored as a candidate biomarker for evaluating IL-13 activity in the airway. We describe the technical performance characteristics of a novel, fully automated immunoassay for the determination of periostin in serum. DESIGN AND METHODS: Limit of blank [LoB], limit of detection [LoD] and limit of quantitation [LoQ], linearity, precision and reproducibility across sites and lots were evaluated according to Clinical and Laboratory Standards Institute guidelines. Interferences and sample stability were also investigated. RESULTS: The pre-specified values for LoB (2ng/mL), LoD (4ng/mL) and LoQ (10ng/mL) were met. The assay was linear throughout the measuring range (10-160ng/mL) with recoveries within ±10% of target at concentrations >30ng/mL and within ±3ng/mL at concentrations ≤30ng/mL. Recovered periostin concentrations were also within ±10% of target in presence of 43 potentially interfering substances and drugs. Samples were stable across various storage conditions and durations (24h at room temperature, 7days at 4°C, 12weeks at -20°C, and 3 freeze/thaw cycles). Repeatability experiments resulted in CVs across samples and controls ranging from 0.9-1.5%. Intermediate precision was 1.2-1.7% and reproducibility including 3 testing sites and 3 reagent lots was 1.7-3.1%. The final assay correlates to the assay version used in previous clinical trials (Pearson's r=0.998, bias at 50ng/mL=1.2%). CONCLUSION: The performance evaluation of the Elecsys® Periostin immunoassay including a multicenter precision analysis demonstrated that the assay is suitable for measuring serum periostin at clinically important concentrations around 50ng/mL.
Asunto(s)
Inmunoensayo/métodos , Biomarcadores/análisis , Moléculas de Adhesión Celular/análisis , Humanos , Reproducibilidad de los ResultadosRESUMEN
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.
Asunto(s)
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
'MegaPlex PCR' is a robust technology for highly multiplexed amplification of specific DNA sequences. It uses target-specific pairs of PCR primers that are physically separated by surface immobilization. Initial surface-based amplification cycles are then coupled to efficient solution-phase PCR using one common primer pair. We demonstrate this method by co-amplifying and genotyping 75 unselected human single-nucleotide polymorphism (SNP) loci.