Dataset Information

Design and Validation of a Custom Next-Generation Sequencing Panel in Pediatric Acute Lymphoblastic Leukemia.

ABSTRACT: The molecular landscape of acute lymphoblastic leukemia (ALL) is highly heterogeneous, and genetic lesions are clinically relevant for diagnosis, risk stratification, and treatment guidance. Next-generation sequencing (NGS) has become an essential tool for clinical laboratories, where disease-targeted panels are able to capture the most relevant alterations in a cost-effective and fast way. However, comprehensive ALL panels assessing all relevant alterations are scarce. Here, we design and validate an NGS panel including single-nucleotide variants (SNVs), insertion-deletions (indels), copy number variations (CNVs), fusions, and gene expression (ALLseq). ALLseq sequencing metrics were acceptable for clinical use and showed 100% sensitivity and specificity for virtually all types of alterations. The limit of detection was established at a 2% variant allele frequency for SNVs and indels, and at a 0.5 copy number ratio for CNVs. Overall, ALLseq is able to provide clinically relevant information to more than 83% of pediatric patients, making it an attractive tool for the molecular characterization of ALL in clinical settings.

SUBMITTER: Gil JV

PROVIDER: S-EPMC10002321 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

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Publications

Design and Validation of a Custom Next-Generation Sequencing Panel in Pediatric Acute Lymphoblastic Leukemia.

Gil José Vicente JV Such Esperanza E Sargas Claudia C Simarro Javier J Miralles Alberto A Pérez Gema G de Juan Inmaculada I Palanca Sarai S Avetisyan Gayane G Santiago Marta M Fuentes Carolina C Fernández José María JM Vicente Ana Isabel AI Romero Samuel S Llop Marta M Barragán Eva E

International journal of molecular sciences 20230223 5

The molecular landscape of acute lymphoblastic leukemia (ALL) is highly heterogeneous, and genetic lesions are clinically relevant for diagnosis, risk stratification, and treatment guidance. Next-generation sequencing (NGS) has become an essential tool for clinical laboratories, where disease-targeted panels are able to capture the most relevant alterations in a cost-effective and fast way. However, comprehensive ALL panels assessing all relevant alterations are scarce. Here, we design and valid ...[more]

PMID: 36901871

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Project description:DNA methylation is the most recognized epigenetic mark that leads to a massive distortion in cancer cells. It has been observed that a large number of DNA aberrant methylation events occur simultaneously in a group of genes, thus providing a growth advantage to the cell in promoting cell differentiation and neoplastic transformation. Due to this reason, methylation profiles have been suggested as promising cancer biomarkers. Here, we designed and performed a first step of validation of a novel targeted next generation sequencing (NGS) panel for methylation analysis, which can simultaneously evaluate the methylation levels at CpG sites of multiple cancer-related genes. The OPERA_MET-A methylation panel was designed using the Ion AmpliSeq™ technology to amplify 155 regions with 125-175 bp mean length and covers a total of 1107 CpGs of 18 cancer-related genes. The performance of the panel was assessed by running commercially available fully methylated and unmethylated control human genomic DNA (gDNA) samples and a variable mixture of them. The libraries were run on Ion Torrent platform and the sequencing output was analyzed using the "methylation_analysis" plugin. DNA methylation calls on both Watson (W) and Crick (C) strands and methylated:unmethylated ratio for each CpG site were obtained. Cell lines, fresh frozen and formalin-fixed paraffin-embedded (FFPE) lung cancer tissues were tested. The OPERA_MET-A panel allows to run a minimum of 6 samples/530 chip to reach an observed mean target depth ≥2,500X (W and C strands) and an average number of mapped reads >750,000/sample. The conversion efficiency, determined by spiking-in unmethylated Lambda DNA into each sample before the bisulfite conversion process, was >97% for all samples. The observed percentage of global methylation for all CpGs was >95% and <5% for fully methylated and unmethylated gDNA samples, respectively, and the observed results for the variable mixtures were in agreement with what was expected. Methylation-specific NGS analysis represents a feasible method for a fast and multiplexed screening of cancer patients by a high-throughput approach. Moreover, it offers the opportunity to construct a more robust algorithm for disease prediction in cancer patients having a low quantity of biological material available.

Dataset Information

Design and Validation of a Custom Next-Generation Sequencing Panel in Pediatric Acute Lymphoblastic Leukemia.

Publications

Design and Validation of a Custom Next-Generation Sequencing Panel in Pediatric Acute Lymphoblastic Leukemia.

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