Dataset Information

A Simple RNA Target Capture NGS Strategy for Fusion Genes Assessment in the Diagnostics of Pediatric B-cell Acute Lymphoblastic Leukemia.

ABSTRACT: Acute lymphoblastic leukemia (ALL) is the most frequent pediatric cancer. Fusion genes are hallmarks of ALL, and they are used as biomarkers for risk stratification as well as targets for precision medicine. Hence, clinical diagnostics pursues broad and comprehensive strategies for accurate discovery of fusion genes. Currently, the gold standard methodologies for fusion gene detection are fluorescence in situ hybridization and polymerase chain reaction; these, however, lack sensitivity for the identification of new fusion genes and breakpoints. In this study, we implemented a simple operating procedure (OP) for detecting fusion genes. The OP employs RNA CaptureSeq, a versatile and effortless next-generation sequencing assay, and an in-house as well as a purpose-built bioinformatics pipeline for the subsequent data analysis. The OP was evaluated on a cohort of 89 B-cell precursor ALL (BCP-ALL) pediatric samples annotated as negative for fusion genes by the standard techniques. The OP confirmed 51 samples as negative for fusion genes, and, more importantly, it identified known (KMT2A rearrangements) as well as new fusion events (JAK2 rearrangements) in the remaining 38 investigated samples, of which 16 fusion genes had prognostic significance. Herein, we describe the OP and its deployment into routine ALL diagnostics, which will allow substantial improvements in both patient risk stratification and precision medicine.

SUBMITTER: Grioni A

PROVIDER: S-EPMC6746019 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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A Simple RNA Target Capture NGS Strategy for Fusion Genes Assessment in the Diagnostics of Pediatric B-cell Acute Lymphoblastic Leukemia.

Grioni Andrea A Fazio Grazia G Rigamonti Silvia S Bystry Vojtech V Daniele Giulia G Dostalova Zuzana Z Quadri Manuel M Saitta Claudia C Silvestri Daniela D Songia Simona S Storlazzi Clelia T CT Biondi Andrea A Darzentas Nikos N Cazzaniga Giovanni G

HemaSphere 20190604 3

Acute lymphoblastic leukemia (ALL) is the most frequent pediatric cancer. Fusion genes are hallmarks of ALL, and they are used as biomarkers for risk stratification as well as targets for precision medicine. Hence, clinical diagnostics pursues broad and comprehensive strategies for accurate discovery of fusion genes. Currently, the gold standard methodologies for fusion gene detection are fluorescence in situ hybridization and polymerase chain reaction; these, however, lack sensitivity for the i ...[more]

PMID: 31723839

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Project description:Background: Global surveillance of viral sequence diversity is needed to keep pace with the constant evolution of HIV. Recent next generation sequencing (NGS) methods have realized the goal of sequencing circulating virus directly from patient specimens. Yet, a simple, universal approach that maximizes sensitivity and sequencing capacity remains elusive. Here we present a novel HIV enrichment strategy to yield near complete genomes from low viral load specimens. Methodology: A non-redundant biotin-labeled probe set (HIV-xGen; n = 652) was synthesized to tile all HIV-1 (groups M, N, O, and P) and HIV-2 (A and B) strains. Illumina Nextera barcoded libraries of either gene-specific or randomly primed cDNA derived from infected plasma were hybridized to probes in a single pool and unbound sequences were washed away. Captured viral cDNA was amplified by Illumina adaptor primers, sequenced on a MiSeq, and NGS reads were demultiplexed for alignment with CLC Bio software. Results: HIV-xGen probes selectively captured and amplified reads spanning the entirety of the HIV phylogenetic tree. HIV sequences clearly present in unenriched libraries of specimens but previously not observed due to high host background levels, insufficient sequencing depth or the extent of multiplexing, were now enriched by >1,000-fold. Thus, xGen selection not only substantially increased the depth of existing sequence, but also extended overall genome coverage by an average of 40%. We characterized 50 new, diverse HIV strains from clinical specimens and demonstrated a viral load cutoff of approximately log 3.5 copies/ml for full length coverage. Genome coverage was <20% for 5/10 samples with viral loads <log 3.5 copies/ml and >90% for 35/40 samples with higher viral loads. Conclusions: Characterization of >20 complete genomes at a time is now possible from a single probe hybridization and MiSeq run. With the versatility to capture all HIV strains and the sensitivity to detect low titer specimens, HIV-xGen will serve as an important tool for monitoring HIV sequence diversity.

Dataset Information

A Simple RNA Target Capture NGS Strategy for Fusion Genes Assessment in the Diagnostics of Pediatric B-cell Acute Lymphoblastic Leukemia.

Publications

A Simple RNA Target Capture NGS Strategy for Fusion Genes Assessment in the Diagnostics of Pediatric B-cell Acute Lymphoblastic Leukemia.

Similar Datasets

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