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. 2019 Jun 13;33(9):2227–2240. doi: 10.1038/s41375-019-0508-7

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — Sensitivity of next-generation sequencing (NGS)-based clonality assessment on lymphoid tissue and lymphoma specimens. a The abundance of IG gene rearrangements and the relative distribution of clonotypes was analyzed using decreasing concentrations of Qubit-quantified input DNA for each multiplex PCR reaction, ranging from 40 to 2.5 ng, of a formalin-fixed paraffin-embedded (FFPE) tonsil specimen (Tonsil2). At 2.5 ng input DNA several IGHV-IGHD-IGHJ sequences showed evidence of pseudoclonality (indicated by circles) or were not detected at all (indicated by arrows). b Two clonal B cell lymphoma specimens (Diag1 and Blym6) were tested in NGS-based clonality analysis using decreasing amount of input DNA (40–1.25 ng). The data as obtained for IGKV-IGKJ gene rearrangement analysis is presented, with detection of the abundant clone even at 1.25 ng input DNA. The green (IGKV5-2/IGKJ2) and orange (IGKV1D-8/IGKJ4) bar of Diag1 represent bi-allelic IGKV-IGKJ gene rearrangements. The green (IGKV1(D)-39/IGKJ2 = J3) and orange (IGKV1(D)-39/IGKJ2) bars of Blym6 represent the same clonotype but ARRest/Interrogate distinguished two variants, since the sequence of the IGKJ gene was assigned differently. The purple, pink, and other colored bars represent additional minor clonotypes