Quality control and quantification in IG/TR next-generation sequencing marker identification: protocols and bioinformatic functionalities by EuroClonality-NGS

Henrik Knecht ,Blanca Scheijen ,Kostas Stamatopoulos ,Van Dongen Jjm ,Michael Svatoň ,David González ,John Moppett ,Eva Froňková ,E Macintyre ,Michaela Kotrová ,Franziska Appelt ,Bystry ,Michael Hummel ,Peter Stewart ,Langerak Aw ,Groenen Pjta ,Jos Rijntjes ,Jeremy Hancock ,Jack Bartram ,Karla Plevová ,Monika Brüggemann ,Christiane Pott ,Simona Songia ,Pál K ,Doris Herrmann ,Frédéric Davi ,Giovanni Cazzaniga ,Adam Krejčí ,Jan Trka ,Ramón García‐Sanz ,Andrea Grioni ,Kamila Stránská ,Tomáš Reigl ,Nikos Darzentas

doi:10.1038/s41375-019-0499-4

Abstract

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.

Highlights

Identification and assessment of clonal immunoglobulin (IG) and T cell receptor (TR) gene rearrangements is a widely used tool for the diagnosis of lymphoid malignancies, and is essential for monitoring minimal residual disease (MRD) [1,2,3,4,5,6].Next-generation sequencing (NGS) of IG/TR gene rearrangements is gaining popularity in clinical laboratories, as it avoids laborious design of patient-specific real-time
Primers are bioinformatically identified in the reads of each of the eight cPT-quality control (QC) tubes of the run and their abundances compared to stored central polytarget QC (cPT-QC) reference results using the test of proportions
We introduce protocols developed within the EuroClonality-next-generation sequencing (NGS) Working Group for QC and quantification in NGS-based IG/TR marker identification

Summary

Introduction

Identification and assessment of clonal immunoglobulin (IG) and T cell receptor (TR) gene rearrangements is a widely used tool for the diagnosis of lymphoid malignancies, and is essential for monitoring minimal residual disease (MRD) [1,2,3,4,5,6].Next-generation sequencing (NGS) of IG/TR gene rearrangements is gaining popularity in clinical laboratories, as it avoids laborious design of patient-specific real-time.

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Conclusion