TAG-CLL, a Novel Tagmentation-Based Approach to Somatic Hypermutation Testing of IGHV Reveals the Weak Points of Both Sanger and Next-Generation Sequencing Methods

Abstract

Somatic hypermutation (SHM) status of IGHV gene, despite being a mature diagnostic biomarker in chronic lymphocytic leukemia (CLL), poses serious methodological problems for molecular laboratories. They may choose between inefficient Sanger sequencing protocols and expensive, recently developed next-generation sequencing-based methods. The performance of both types of methods seemed incomparable, and concerted validation of different protocols between laboratories was inconsiderable. Here, a new tagmentation-based approach to sequencing of IGHV locus is presented, which is agnostic to the amplification protocol used and enables direct comparison of the amplicons and libraries dedicated to different platforms (Sanger, IonTorrent, and Illumina). To demonstrate its potential, the 12 associated molecular diagnostics laboratories were asked to amplify an artificially prepared oligoclonal DNA sample containing a near-equimolar mixture of IGHV clonotypes from six different classes. The PCR products collected from laboratories were then tagmented and sequenced according to the common TAG-CLL workflow. The productivity, degree of germline identity, and SHM status concordance between laboratories have been analyzed. Moreover, systematic biases toward uneven amplification of different clonotypes and the prevalence of accidental artifacts in vitro and in silico have been evaluated, providing a framework for future validation of IGHV SHM methods and next-generation sequencing immunoinformatic pipeline benchmarking.