RESUMO
Chromosomal rearrangements are important drivers in cancer, and their robust detection is essential for diagnosis, prognosis, and treatment selection, particularly for bone and soft tissue tumors. Current diagnostic methods are hindered by limitations, including difficulties with multiplexing targets and poor quality of RNA. A novel targeted DNA-based next-generation sequencing method, formalin-fixed, paraffin-embedded-targeted locus capture (FFPE-TLC), has shown advantages over current diagnostic methods when applied on FFPE lymphomas, including the ability to detect novel rearrangements. We evaluated the utility of FFPE-TLC in bone and soft tissue tumor diagnostics. FFPE-TLC sequencing was successfully applied on noncalcified and decalcified FFPE samples (n = 44) and control samples (n = 19). In total, 58 rearrangements were identified in 40 FFPE tumor samples, including three previously negative samples, and none was identified in the FFPE control samples. In all five discordant cases, FFPE-TLC could identify gene fusions where other methods had failed due to either detection limits or poor sample quality. FFPE-TLC achieved a high specificity and sensitivity (no false positives and negatives). These results indicate that FFPE-TLC is applicable in cancer diagnostics to simultaneously analyze many genes for their involvement in gene fusions. Similar to the observation in lymphomas, FFPE-TLC is a good DNA-based alternative to the conventional methods for detection of rearrangements in bone and soft tissue tumors.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Neoplasias de Tecidos Moles , Humanos , Inclusão em Parafina/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , DNA/genética , Formaldeído , Neoplasias de Tecidos Moles/diagnóstico , Neoplasias de Tecidos Moles/genética , Fusão Gênica , Tecnologia , Fixação de TecidosRESUMO
Targeted locus amplification (TLA) allows for the detection of all genetic variation (including structural variation) in a genomic region of interest. As TLA is based on proximity ligation, variants can be linked to each other, thereby enabling allelic phasing and the generation of haplotypes. This allows for the study of genetic variants in an allele-specific manner. Here, we provide a step-by-step protocol for TLA sample preparation and a complete bioinformatics pipeline for the allelic phasing of TLA data. Additionally, to illustrate the protocol, we show the ability of TLA to re-sequence and haplotype the complete cystic fibrosis transmembrane (CFTR) gene (> 200 kb in size) from patient-derived intestinal organoids.
Assuntos
Fibrose Cística , Genômica , Humanos , Haplótipos/genética , Genômica/métodos , Alelos , Fibrose Cística/genéticaRESUMO
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
We present TaxPhlAn, a new method and bioinformatics pipeline for design and analysis of single-locus sequence typing (SLST) markers to type and profile bacteria beyond the species-level in a complex microbial community background. TaxPhlAn can be applied to any group of phylogenetically-related bacteria, provided reference genomes are available. As TaxPhlAn requires the SLST targets identified to fit the phylogenetic pattern as determined through comprehensive evolutionary reconstruction of input genomes, TaxPhlAn allows for the identification and phylogenetic inference of new biodiversity. Here, we present a clinically relevant case study of high-resolution Staphylococcus profiling on skin of atopic dermatitis (AD) patients. We demonstrate that SLST enables profiling of cutaneous Staphylococcus members at (sub)species level and provides higher resolution than current 16S-based techniques. With the higher discriminative ability provided by our approach, we further show that the presence of Staphylococcus capitis on the skin together with Staphylococcus aureus associates with AD disease.
Assuntos
Bactérias/genética , Técnicas de Tipagem Bacteriana/métodos , Biologia Computacional/métodos , Genes Bacterianos/genética , Microbiota/genética , Bactérias/classificação , Dermatite Atópica/microbiologia , Feminino , Humanos , Masculino , Filogenia , Pele/microbiologia , Pele/patologia , Especificidade da Espécie , Infecções Estafilocócicas/microbiologia , Staphylococcus/classificação , Staphylococcus/genética , Staphylococcus/fisiologia , Fluxo de TrabalhoRESUMO
Lactobacillus delbrueckii subsp. bulgaricus LBB.B5 originates from homemade Bulgarian yogurt and was selected for its ability to form a strong association with Streptococcus thermophilus The genome sequence will facilitate elucidating the genetic background behind the contribution of LBB.B5 to the taste and aroma of yogurt and its exceptional protocooperation with S. thermophilus.