Comprehensive translocation and clonality detection in lymphoproliferative disorders by next-generation sequencing.
Ontology highlight
ABSTRACT:
SUBMITTER: Wren D
PROVIDER: S-EPMC5286955 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Balanced chromosome rearrangements (BCRs) can cause genetic diseases by disrupting or inactivating specific genes, and the characterization of breakpoints in disease-associated BCRs has been instrumental in the molecular elucidation of a wide variety of genetic disorders. However, mapping chromosome breakpoints using traditional methods, such as in situ hybridization with fluorescent dye-labeled bacterial artificial chromosome clones (BAC-FISH), is rather laborious and time-consuming. In addition, the resolution of BAC-FISH is often insufficient to unequivocally identify the disrupted gene. To overcome these limitations, we have performed shotgun sequencing of flow-sorted derivative chromosomes using "next-generation" (Illumina/Solexa) multiplex sequencing-by-synthesis technology. As shown here for three different disease-associated BCRs, the coverage attained by this platform is sufficient to bridge the breakpoints by PCR amplification, and this procedure allows the determination of their exact nucleotide positions within a few weeks. Its implementation will greatly facilitate large-scale breakpoint mapping and gene finding in patients with disease-associated balanced translocations.
| S-EPMC2493403 | biostudies-literature
Project description:Clonality analysis of viral vector-transduced cell populations represents a convincing approach to dissect the physiology of tissue and organ regeneration, to monitor the fate of individual gene-corrected cells in vivo, and to assess vector biosafety. With the decoding of mammalian genomes and the introduction of next-generation sequencing technologies, the demand for automated bioinformatic analysis tools that can rapidly process and annotate vector integration sites is rising. Here, we provide a publicly accessible, graphical user interface-guided automated bioinformatic high-throughput integration site analysis pipeline. Its performance and key features are illustrated on pyrosequenced linear amplification-mediated PCR products derived from one patient previously enrolled in the first lentiviral vector clinical gene therapy study. Analysis includes trimming of vector genome junctions, alignment of genomic sequence fragments to the host genome for the identification of integration sites, and the annotation of nearby genomic elements. Most importantly, clinically relevant features comprise the determination of identical integration sites with respect to different time points or cell lineages, as well as the retrieval of the most prominent cell clones and common integration sites. The resulting output is summarized in tables within a convenient spreadsheet and can be further processed by researchers without profound bioinformatic knowledge.
| S-EPMC4015219 | biostudies-literature
Project description:Balanced chromosome rearrangements (BCRs) can cause genetic diseases by disrupting or inactivating specific genes, and the characterisation of breakpoints in disease-associated BCRs has been instrumental in the molecular elucidation of a wide variety of genetic disorders. However, mapping chromosome breakpoints using traditional methods, such as in situ hybridization with fluorescent dye-labeled bacterial artificial chromosome clones (BAC-FISH), is rather laborious and time consuming. In addition, the resolution of BAC-FISH is often insufficient to unequivocally identify the disrupted gene. To overcome these limitations, we have performed shotgun sequencing of flow-sorted derivative chromosomes using ânext generationâ (Solexa/Illumina) multiplex sequencing-by-synthesis technology. As shown here for three different disease-associated BCRs, the coverage attained by this platform is sufficient to bridge the breakpoints by PCR amplification, and this procedure allows to determine their exact nucleotide positions within few weeks. Its implementation will greatly facilitate large-scale breakpoint mapping and gene finding in patients with disease-associated balanced translocations. Array CGH was performed in three carriers of balanced translocations to exclude DNA copy number changes.
2010-05-22 | E-GEOD-10115 | biostudies-arrayexpress
Project description:Bloodstream infection (BSI) is a severe complication in immunocompromised patients. Next-generation sequencing (NGS) allows us to analyze comprehensively and quantitatively all microorganisms present in a clinical sample. Thirty-five pediatric patients (12 with BSI and 23 with suspected BSI/negative blood culture) were enrolled. Plasma/serum samples were used for sequencing and the results were compared with those from blood culture. Sequencing reads of bacteria isolated in blood culture were identified by NGS in all plasma/serum samples at disease onset. Bacteria isolated in blood culture were identical to the dominant bacteria by NGS in 8 of 12 patients. Bacterial reads per million reads of the sequence depth (BR) > 200 and relative importance values of the dominant bacteria (P1)?>?0.5 were employed to determine causative pathogens. Causative pathogens were detected using these criteria in 7 of 12 patients with BSI. Additionally, causative bacteria were detected in the plasma/serum at 7 days before disease onset in two patients with catheter-related BSI. Causative pathogens, including virus, were identified in three patients with suspected BSI. Lastly, a total of 62 resistance genes were detected by NGS. In conclusion, NGS is a new method to identify causative microorganisms in BSI and may predict BSI in some patients.
| S-EPMC5830625 | biostudies-literature
Project description:NGPS is a method for de-novo, full-length protein sequencing in high throughput. The method is based on cleavage of the protein at semi-random sites by microwave-assisted acid hydrolysis (MAAH), enrichment of LC-MS/MS amenable peptides from the hydrolysate by solid-phase-extraction, LC-MS/MS analysis, de-novo long peptide tag sequencing of resulting peptides and assembly of peptide tags into consensus contigs.
2017-04-03 | PXD003804 | Pride
Project description:Next-generation DNA sequencing promises to revolutionize clinical medicine and basic research. However, while this technology has the capacity to generate hundreds of billions of nucleotides of DNA sequence in a single experiment, the error rate of ~1% results in hundreds of millions of sequencing mistakes. These scattered errors can be tolerated in some applications but become extremely problematic when "deep sequencing" genetically heterogeneous mixtures, such as tumors or mixed microbial populations. To overcome limitations in sequencing accuracy, we have developed a method termed Duplex Sequencing. This approach greatly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors result in mutations in only one strand and can thus be discounted as technical error. We determine that Duplex Sequencing has a theoretical background error rate of less than one artifactual mutation per billion nucleotides sequenced. In addition, we establish that detection of mutations present in only one of the two strands of duplex DNA can be used to identify sites of DNA damage. We apply the method to directly assess the frequency and pattern of random mutations in mitochondrial DNA from human cells.
| S-EPMC3437896 | biostudies-other
Project description:Tumors are characterized by properties of genetic instability, heterogeneity, and significant oligoclonality. Elucidating this intratumoral heterogeneity is challenging but important. In this study, we propose a framework, BubbleTree, to characterize the tumor clonality using next generation sequencing (NGS) data. BubbleTree simultaneously elucidates the complexity of a tumor biopsy, estimating cancerous cell purity, tumor ploidy, allele-specific copy number, and clonality and represents this in an intuitive graph. We further developed a three-step heuristic method to automate the interpretation of the BubbleTree graph, using a divide-and-conquer strategy. In this study, we demonstrated the performance of BubbleTree with comparisons to similar commonly used tools such as THetA2, ABSOLUTE, AbsCN-seq and ASCAT, using both simulated and patient-derived data. BubbleTree outperformed these tools, particularly in identifying tumor subclonal populations and polyploidy. We further demonstrated BubbleTree's utility in tracking clonality changes from patients' primary to metastatic tumor and dating somatic single nucleotide and copy number variants along the tumor clonal evolution. Overall, the BubbleTree graph and corresponding model is a powerful approach to provide a comprehensive spectrum of the heterogeneous tumor karyotype in human tumors. BubbleTree is R-based and freely available to the research community (https://www.bioconductor.org/packages/release/bioc/html/BubbleTree.html).
| S-EPMC4770205 | biostudies-literature
Project description:New advances in genomic technology are being introduced at a greater speed and are revolutionizing the field of genetics for both complex and Mendelian diseases. For instance, during the past few years, genome-wide association studies (GWAS) have identified a large number of significant associations between genomic loci and movement disorders such as Parkinson's disease and progressive supranuclear palsy. GWAS are carried out through the use of high-throughput SNP genotyping arrays, which are also used to perform linkage analyses in families previously considered statistically underpowered for genetic analyses. In inherited movement disorders, using this latter technology, it has repeatedly been shown that mutations in a single gene can lead to different phenotypes, while the same clinical entity can be caused by mutations in different genes. This is being highlighted with the use of next-generation sequencing technologies and leads to the search for genes or genetic modifiers that contribute to the phenotypic expression of movement disorders. Establishing an accurate genome-epigenome-phenotype relationship is becoming a major challenge in the post-genomic research that should be facilitated through the implementation of both functional and cellular analyses.
| S-EPMC3351011 | biostudies-literature
Project description:The availability of Bruton tyrosine kinase (BTK) inhibitors has brought about a paradigm shift in the treatment of patients with B-cell lymphomas and chronic lymphocytic leukemia. BTK was clinically validated as a target by the efficacy of the first-in-class inhibitor ibrutinib. The extended survival conferred by BTK inhibitors has brought long-term tolerability to the foreground. To minimize toxicities thought to be attributable to off-target kinase inhibition, a next generation of BTK inhibitors with greater selectivity was developed. In the United States, zanubrutinib, a next-generation BTK inhibitor, has been approved for treating adults with mantle cell lymphoma who have received at least one prior therapy, for adults with Waldenström macroglobulinemia, and for adults with relapsed or refractory marginal zone lymphoma who have received at least one anti-CD20-based therapy. Because few head-to-head comparative trials of BTK inhibitors have so far been reported, no BTK 'inhibitor of choice' can be identified. Zanubrutinib has promising efficacy in its approved indications and appears to have reduced cardiac toxicities, particularly atrial fibrillation, which may influence the choice of BTK inhibitor treatment by prescribers. Further studies are needed to inform on optimal treatment sequencing of zanubrutinib and its combination with other agents. Here, we summarize existing clinical evidence for its efficacy and safety in mantle cell lymphoma, Waldenström macroglobulinemia, marginal zone lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma, and other B-lymphoproliferative indications.Plain language summaryZanubrutinib is a drug that was shown to effectively treat cancer of B cells without causing excessive serious side effects Patients with certain B-cell malignancies (cancers of white blood cells) benefit from treatment with Bruton tyrosine kinase (BTK) inhibitors, drugs that block the BTK protein and keep cancer from growing and spreading. Patients experience extended survival with ibrutinib, the first-generation BTK inhibitor approved by US Food and Drug Administration (FDA); however, one in five patients quit treatment because of harmful side effects. Ibrutinib-related side effects such as increased risk of bleeding, atrial fibrillation (abnormal heart rhythm), and high blood pressure are thought to be caused by ibrutinib blocking other proteins besides the intended target protein BTK. To reduce these side effects, zanubrutinib, a next-generation BTK inhibitor, was designed to block BTK more specifically than ibrutinib. Results of clinical studies on zanubrutinib treatment appear promising in patients with several types of B-cell malignancies, including mantle cell lymphoma (MCL), Waldenström macroglobulinemia (WM), marginal zone lymphoma (MZL), chronic lymphocytic leukemia, and small lymphocytic lymphoma. There are not yet enough clinical data to determine which BTK inhibitor is most effective in treating B-cell malignancies without causing harmful side effects. Early data from the phase 3 ALPINE clinical study suggest that zanubrutinib works better than ibrutinib, and fewer patients experience side effects and quit treatment. Zanubrutinib is currently approved for use for treatment of adult patients with MCL who have received at least one prior therapy, for adults with WM, and for adults with MZL who have received at least one anti-CD20-based therapy.
| S-EPMC9150264 | biostudies-literature
Project description:The interplay between tumor heterogeneity and microenvironmental factors is a critical mechanism for clonal selection in leukemia. Evidence of unique clonal capacities to engraft within patient-derived xenograft (PDX) models suggests that intrapatient genetic architecture may be defined by functional differences at the clonal level. However, methods to detect functional differences assigned to genetically defined clones remain limited. Here, we describe a scalable method to directly measure the functional properties of clones within the same leukemia patient by coupling intracellular flow cytometry and next-generation sequencing (NGS). We provide proof of concept utilizing primary chronic myelmonocytic leukemia (CMML) samples and granulocyte-macrophage colony stimulating factor (GM-CSF) to elucidate the interaction between tumor heterogeneity and microenvironmental factors. Mixtures of human leukemia cell lines, with known response to GM-CSF, were used to validate the accuracy of our methodology. Using this approach, we confirm that our method is capable of discriminating GM-CSF sensitive cell lines, identifies somatic variants in primary leukemia samples, and resolves functional clonal architecture in an illustrative patient. Taken together, our data describes a novel method to determine intrapatient functional clonal heterogeneity and provides proof-of-concept for future investigation aimed at elucidating the clinical relevance of functional clonal differences.
| S-EPMC7771351 | biostudies-literature