RESUMO
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.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Linfoma de Células B/genética , Linfoma não Hodgkin/genética , Inclusão em Parafina/métodos , Fixação de Tecidos/métodos , Translocação Genética , Biologia Computacional/métodos , Rearranjo Gênico , Genes bcl-2/genética , Genes myc/genética , Humanos , Hibridização in Situ Fluorescente/métodos , Linfoma de Células B/diagnóstico , Linfoma não Hodgkin/diagnóstico , Proteínas Proto-Oncogênicas c-bcl-6/genética , Reprodutibilidade dos Testes , Estudos Retrospectivos , Sensibilidade e EspecificidadeRESUMO
In recent years, technologies capable of analyzing single cells have emerged that are transforming many fields of biological research. Herein we report how DNA-functionalized hydrogel beads can serve as a matrix to capture mRNA from lysed single cells. mRNA quantification free of pre-amplification bias is ensured by using padlock probes and rolling circle amplification followed by hybridization with fluorescent probes. The number of transcripts in individual cells is assessed by simply counting fluorescent dots inside gel beads. The method extends the potential of existing techniques and provides a general platform for capturing molecules of interest from single cells.