RESUMEN
In routine diagnostic pathology, cancer biopsies are preserved by formalin-fixed, paraffin-embedding (FFPE) procedures for examination of (intra-) cellular morphology. Such procedures inadvertently induce DNA fragmentation, which compromises sequencing-based analyses of chromosomal rearrangements. Yet, rearrangements drive many types of hematolymphoid malignancies and solid tumors, and their manifestation is instructive for diagnosis, prognosis, and treatment. Here, we present FFPE-targeted locus capture (FFPE-TLC) for targeted sequencing of proximity-ligation products formed in FFPE tissue blocks, and PLIER, a computational framework that allows automated identification and characterization of rearrangements involving selected, clinically relevant, loci. FFPE-TLC, blindly applied to 149 lymphoma and control FFPE samples, identifies the known and previously uncharacterized rearrangement partners. It outperforms fluorescence in situ hybridization (FISH) in sensitivity and specificity, and shows clear advantages over standard capture-NGS methods, finding rearrangements involving repetitive sequences which they typically miss. FFPE-TLC is therefore a powerful clinical diagnostics tool for accurate targeted rearrangement detection in FFPE specimens.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Linfoma de Células B/genética , Linfoma no Hodgkin/genética , Adhesión en Parafina/métodos , Fijación del Tejido/métodos , Translocación Genética , Biología Computacional/métodos , Reordenamiento Génico , Genes bcl-2/genética , Genes myc/genética , Humanos , Hibridación Fluorescente in Situ/métodos , Linfoma de Células B/diagnóstico , Linfoma no Hodgkin/diagnóstico , Proteínas Proto-Oncogénicas c-bcl-6/genética , Reproducibilidad de los Resultados , Estudios Retrospectivos , Sensibilidad y EspecificidadRESUMEN
Next-generation sequencing (NGS) is rapidly becoming the method of choice for mutation analysis in both research and diagnostics. The benefit of targeted NGS compared to whole-genome and whole-exome sequencing is that smaller amounts of input material can be used as well as qualitatively suboptimal tissue samples, like formalin-fixed, paraffin-embedded archival tissue.Here, we describe the protocol for targeted next-generation sequencing using the Ion Torrent PGM platform in combination with Ion Ampliseq NGS gene panels for formalin-fixed, paraffin-embedded tissues. Both the manual and the automated workflow are described as well as the bioinformatics for data analysis.
Asunto(s)
Análisis Mutacional de ADN/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Adhesión en Parafina , Fijación del Tejido , Biología Computacional/métodos , Formaldehído , Humanos , Neoplasias/genéticaRESUMEN
Despite developments in targeted gene sequencing and whole-genome analysis techniques, the robust detection of all genetic variation, including structural variants, in and around genes of interest and in an allele-specific manner remains a challenge. Here we present targeted locus amplification (TLA), a strategy to selectively amplify and sequence entire genes on the basis of the crosslinking of physically proximal sequences. We show that, unlike other targeted re-sequencing methods, TLA works without detailed prior locus information, as one or a few primer pairs are sufficient for sequencing tens to hundreds of kilobases of surrounding DNA. This enables robust detection of single nucleotide variants, structural variants and gene fusions in clinically relevant genes, including BRCA1 and BRCA2, and enables haplotyping. We show that TLA can also be used to uncover insertion sites and sequences of integrated transgenes and viruses. TLA therefore promises to be a useful method in genetic research and diagnostics when comprehensive or allele-specific genetic information is needed.
Asunto(s)
Genómica/métodos , Haplotipos/genética , Modelos Genéticos , Técnicas de Amplificación de Ácido Nucleico/métodos , Análisis de Secuencia de ADN/métodos , Fusión Génica/genética , Genes BRCA1 , Genes BRCA2 , Sitios Genéticos/genética , Humanos , Neoplasias/genética , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
Chromosome conformation capture (3C) technology and its genome-wide derivatives have revolutionized our knowledge on chromatin folding and nuclear organization. 4C-seq Technology combines 3C principles with high-throughput sequencing (4C-seq) to enable for unbiased genome-wide screens for DNA contacts made by single genomic sites of interest. Here, we discuss in detail the design, application, and data analysis of 4C-seq experiments. Based on many hundreds of different 4C-seq experiments, we define criteria to assess data quality and show how different restriction enzymes and cross-linking conditions affect results. We describe in detail the mapping strategy of 4C-seq reads and show advanced strategies for data analysis.
Asunto(s)
Cromatina/química , Mapeo Cromosómico/métodos , ADN/química , Análisis de Secuencia de ADN/métodos , Estadística como Asunto/métodos , Ensamble y Desensamble de Cromatina , Reactivos de Enlaces Cruzados , ADN/genética , Enzimas de Restricción del ADN/química , Formaldehído/química , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Conformación de Ácido Nucleico , Reacción en Cadena de la Polimerasa/métodos , Globinas beta/química , Globinas beta/genéticaRESUMEN
BACKGROUND: Complex chromosomal rearrangements (CCR) are rare cytogenetic findings that are difficult to karyotype by conventional cytogenetic analysis partially because of the relative low resolution of this technique. High resolution genotyping is necessary in order to identify cryptic imbalances, for instance near the multiple breakpoints, to explain the abnormal phenotype in these patients. We applied several molecular techniques to elucidate the complexity of the CCRs of two adult patients with abnormal phenotypes. RESULTS: Multicolour fluorescence in situ hybridization (M-FISH) showed that in patient 1 the chromosomes 1, 10, 15 and 18 were involved in the rearrangement whereas for patient 2 the chromosomes 5, 9, 11 and 13 were involved. A 250 k Nsp1 SNP-array analysis uncovered a deletion in chromosome region 10p13 for patient 1, harbouring 17 genes, while patient 2 showed no pathogenic gains or losses. Additional FISH analysis with locus specific BAC-probes was performed, leading to the identification of cryptic interstitial structural rearrangements in both patients. CONCLUSION: Application of M-FISH and SNP-array analysis to apparently balanced CCRs is useful to delineate the complex chromosomal rearrangement in detail. However, it does not always identify cryptic imbalances as an explanation for the abnormal phenotype in patients with a CCR.