Figure 1.

Workflow for MRD assessment by RQ-PCR and high-throughput sequencing targeting the clonal immunoglobulin heavy chain gene (IGH) rearrangement. (A) Consensus PCR for assessment of clonal IGH gene rearrangements is followed by Sanger sequencing to identify the clonal V_H-N-D_H-N-J_H region for each patient. Allele-specific primers are used for sensitive quantification of residual tumor cells by RQ-PCR. (B) An amplicon-sequencing strategy is used for identification of clonal IGH gene rearrangement in a 2-step PCR approach, in which the first PCR is performed by using multiplex gene-specific primers tailed with a universal linker sequence. The universal tailed amplicons can be used for second-round PCR, in which next-generation sequencing platform-specific adapters can be introduced, depending on the platform used for sequencing.