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. 2021 Jun 18;11:12880. doi: 10.1038/s41598-021-91974-x

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© The Author(s) 2021

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Colorectal dMMR/MSI tumor (case 27) with concordant results between IHC and all three molecular methods. Hematoxylin eosin stain (a) of the tumor with classic morphology of colorectal adenocarcinoma with combined nuclear loss of expression of MLH1 (b) and PMS2 (d) and preserved nuclear staining for MSH2 (c) and MSH6 (e). Idylla MSI assay (f) indicates 5/7 loci MSI resulting in a global MSI-high score (Idylla Explore software v.2.5.1294). Illustrative indel distribution plot of the FLT1 locus with clearly higher numbers of integrated peaks. An indel length peak is integrated by mSINGS and counted when the fraction of aligned reads per indel length is higher than 5% of total reads for the entire locus (peak value, > 0.05, threshold for peak integration indicated by red line (plot using Python v.3.7 and Matplotlib v.3.3.4). Here the number of integrated peaks on the FLT1 locus in the test sample (dark blue, 8 peaks above threshold) is higher than number of peaks in the trained baseline (light blue, 6 peaks) thus binary calling this locus as MSI. Representative fragment length distributions as measured by PCR (GeneMapper v. 4.0 analysis software, Applied Biosystems). (g) and manually interpreted as MSI by a trained observer. (h) PCR results for 8 microsatellite markers clearly show a wide range of alleles for all loci.