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. 2022 Mar 28;35(8):1022–1033. doi: 10.1038/s41379-022-01058-y

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© The Author(s) 2022

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 — A Cartoon showing the rationale for designing an additional FLI1-targeting primer and probe. The initial design is indicated by black colored primers and probes and the new design is indicated by a red primer and probe. The new design results in partner primers and probes binding less than 130 base pairs from each other when the targeted fusion transcript breakpoint is between exon 7 of EWSR1 and exon 6 of FLI1. In this way, complementary primers and probes can successfully bind to cDNA made from RNA strands with a fragment length of 200 base pairs or less to accommodate fusion transcript. B The addition of the new FLI1-targeting primer and probe to the initial tiered multiplexed primer and probe pools does not disrupt detection of the expected fusion in samples that were previously positive and (C) improves the fusion detection for two EWSR1-FLI1 positive tumor biopsy samples as intended. It was still not possible to detect the EWSR1-ERG fusion transcript for MRD0011 sample despite a reduction of the amplicon size from 281 bases to 187 bases.