Amplicon-based next-generation sequencing (NGS) of immunoglobulin (IG) and T-cell receptor (TR) gene rearrangements for clonality assessment, marker identification and quantification of minimal residual disease (MRD) in lymphoid neoplasms has been the focus of intense research, development and application. However, standardization and validation in a scientifically controlled multicentre setting is still lacking. Therefore, IG/TR assay development and design, including bioinformatics, was performed within the EuroClonality-NGS working group and validated for MRD marker identification in acute lymphoblastic leukaemia (ALL). Five EuroMRD ALL reference laboratories performed IG/TR NGS in 50 diagnostic ALL samples, and compared results with those generated through routine IG/TR Sanger sequencing. A central polytarget quality control (cPT-QC) was used to monitor primer performance, and a central in-tube quality control (cIT-QC) was spiked into each sample as a library-specific quality control and calibrator. NGS identified 259 (average 5.2/sample, range 0-14) clonal sequences vs. Sanger-sequencing 248 (average 5.0/sample, range 0-14). NGS primers covered possible IG/TR rearrangement types more completely compared with local multiplex PCR sets and enabled sequencing of bi-allelic rearrangements and weak PCR products. The cPT-QC showed high reproducibility across all laboratories. These validated and reproducible quality-controlled EuroClonality-NGS assays can be used for standardized NGS-based identification of IG/TR markers in lymphoid malignancies.

• MeSH
• akutní lymfatická leukemie genetika   MeSH
• genetické markery genetika   MeSH
• genová přestavba T-lymfocytů genetika   MeSH
• geny pro imunoglobuliny genetika   MeSH
• geny TcR genetika   MeSH
• imunoglobuliny genetika   MeSH
• lidé MeSH
• receptory antigenů T-buněk genetika   MeSH
• referenční standardy MeSH
• rekombinace genetická genetika   MeSH
• reprodukovatelnost výsledků MeSH
• reziduální nádor genetika   MeSH
• výpočetní biologie metody   MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• genetické markery MeSH
• imunoglobuliny MeSH
• receptory antigenů T-buněk MeSH

Assessment of clonality, marker identification and measurement of minimal residual disease (MRD) of immunoglobulin (IG) and T cell receptor (TR) gene rearrangements in lymphoid neoplasms using next-generation sequencing (NGS) is currently under intensive development for use in clinical diagnostics. So far, however, there is a lack of suitable quality control (QC) options with regard to standardisation and quality metrics to ensure robust clinical application of such approaches. The EuroClonality-NGS Working Group has therefore established two types of QCs to accompany the NGS-based IG/TR assays. First, a central polytarget QC (cPT-QC) is used to monitor the primer performance of each of the EuroClonality multiplex NGS assays; second, a standardised human cell line-based DNA control is spiked into each patient DNA sample to work as a central in-tube QC and calibrator for MRD quantification (cIT-QC). Having integrated those two reference standards in the ARResT/Interrogate bioinformatic platform, EuroClonality-NGS provides a complete protocol for standardised IG/TR gene rearrangement analysis by NGS with high reproducibility, accuracy and precision for valid marker identification and quantification in diagnostics of lymphoid malignancies.

• MeSH
• genetické markery genetika   MeSH
• genová přestavba genetika   MeSH
• imunoglobuliny genetika   MeSH
• lidé MeSH
• receptory antigenů T-buněk genetika   MeSH
• reprodukovatelnost výsledků MeSH
• reziduální nádor genetika   MeSH
• řízení kvality MeSH
• výpočetní biologie metody   MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• genetické markery MeSH
• imunoglobuliny MeSH
• receptory antigenů T-buněk MeSH

MOTIVATION: The study of immunoglobulins and T cell receptors using next-generation sequencing has finally allowed exploring immune repertoires and responses in their immense variability and complexity. Unsurprisingly, their analysis and interpretation is a highly convoluted task. RESULTS: We thus implemented ARResT/Interrogate, a web-based, interactive application. It can organize and filter large amounts of immunogenetic data by numerous criteria, calculate several relevant statistics, and present results in the form of multiple interconnected visualizations. AVAILABILITY AND IMPLEMENTATION: ARResT/Interrogate is implemented primarily in R, and is freely available at http://bat.infspire.org/arrest/interrogate/ CONTACT: nikos.darzentas@gmail.com SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

• MeSH
• genetická variace MeSH
• imunogenetika metody   MeSH
• imunoglobuliny genetika   MeSH
• lidé MeSH
• receptory antigenů T-buněk genetika   metabolismus   MeSH
• software * MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• imunoglobuliny MeSH
• receptory antigenů T-buněk MeSH