ABSTRACT
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.
Subject(s)
High-Throughput Nucleotide Sequencing/methods , Lymphoma, B-Cell/genetics , Lymphoma, Non-Hodgkin/genetics , Paraffin Embedding/methods , Tissue Fixation/methods , Translocation, Genetic , Computational Biology/methods , Gene Rearrangement , Genes, bcl-2/genetics , Genes, myc/genetics , Humans , In Situ Hybridization, Fluorescence/methods , Lymphoma, B-Cell/diagnosis , Lymphoma, Non-Hodgkin/diagnosis , Proto-Oncogene Proteins c-bcl-6/genetics , Reproducibility of Results , Retrospective Studies , Sensitivity and SpecificityABSTRACT
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.
Subject(s)
Genomics/methods , Haplotypes/genetics , Models, Genetic , Nucleic Acid Amplification Techniques/methods , Sequence Analysis, DNA/methods , Gene Fusion/genetics , Genes, BRCA1 , Genes, BRCA2 , Genetic Loci/genetics , Humans , Neoplasms/genetics , Polymorphism, Single Nucleotide/geneticsABSTRACT
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.