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. 2021 Jun 7;12:3361. doi: 10.1038/s41467-021-23695-8

Fig. 5. FFPE-TLC vs. other state of the art methods.

Fig. 5

A Comparison of FISH, Capture-NGS and FFPE-TLC results showing rearrangements identified in MYC, BCL2, and BCL6 genes across 19 samples. Each circle is a sample that is analyzed for rearrangements in a particular gene. Filled-in circles indicate correspondence with FISH diagnosis and empty (red) circles indicate discordance with FISH diagnosis. B Example of false-negative call by Capture-NGS that was successfully identified by FFPE-TLC. It turned out that Capture-NGS had missed the rearrangement because the region around the breakpoint (red arrowhead) lacked coverage and therefore, the breakpoint could not be identified for sample F190. In contrast as shown in the butterfly plot, rearrangement identification by FFPE-TLC is fusion-read independent and therefore could correctly identify the rearrangement with high confidence (z-score = 82.4). C FFPE-TLC capabilities in detecting translocations even if breakpoints occur far away from the probed (targeted) regions. Each plot demonstrates this ability for a particular gene for two samples, from left to right: BCL2-IGH (shown for F46 and F73), BCL6-IGL (shown for F37 and F45), and MYC-IGH (shown for F50 and F59). The X-axis in each plot indicates the minimum distance between the last probe and the breakpoint position. The Y-axis shows enrichment scores that are computed by PLIER. In all tested cases, PLIER confidently identified the translocation even when the probes are located 50 kb away from the breakpoint. D Diagram showing the fraction of breakpoint sequences from this study that cannot be mapped uniquely on the reference sequence at varying read lengths. For example, even with 60 nucleotides, 5% of FFPE-TLC identified rearrangements would be missed by typical NGS capture methods due to unmappability of the captured sequence. E Schematic view of false-positive call by Capture-NGS in F189 sample. In this case, Capture-NGS identified reads that were spanning the breakpoint and linked the MYC locus to the X chromosome. In contrast, no rearrangement was identified by FFPE-TLC for sample F189. By performing PCR using primers on chromosome X and sequencing, we could successfully explain the event and confirm the insertion of a 240 bp fragment from chromosome 8 into chromosome X.