Fig. 1. Overview of FFPE-TLC and an example of the identified rearrangements.

A Schematic overview of the FFPE-TLC workflow. (1) Through sample fixation, spatially proximal sequences (red) are preferentially cross-linked. Next, paraffin is removed and the sample section is permeabilized to allow enzymes to access the DNA. (2) The DNA is fragmented using NlaIII and then (3) ligated, which results in concatenates of co-localizing DNA fragments. (4) After crosslink reversal and DNA purification, (5) the DNA is subjected to next-generation sequencing library preparation. (6) Sequences of interest are enriched using hybrid capture probes. (7) The prepared library is paired-end Illumina sequenced. B Genome-wide coverage of fragments retrieved from a typical FFPE-TLC experiment targeting MYC, BCL2, and BCL6. Shown in blue is the coverage seen at the (±5 Mb) genomic intervals targeted by the capture probes. The rearranged region to the MYC gene (in green) is identified by the concentration of fragments clustered around the GRHPR gene (chr9:31mb–42mb), shown in red. C The probe sets used in FFPE-TLC not only retrieve the probe-complementary genomic sequences (in blue), but also megabases of its flanking sequences (i.e., the proximity-ligation products), shown for MYC (pink), BCL2 (brown), and BCL6 (orange). In case of a rearrangement (MYC-GRHPR in this case), the corresponding capture probes also retrieve fragments originating from the rearrangement partner (GRHPR, in red). This is not the case for regions that do not harbor any rearrangement (e.g., BCL2 in brown or BCL6 in orange), as shown for the GRHPR locus.