Supplementary Figure 6: Validation of multi-way interactions detected by Tri-C.

a, C-Trap is a novel 3C approach, which can analyze multi-way interactions occurring simultaneously with an interaction between two fragments of interest. It uses long-range PCR amplification with primers targeting these two fragments to enrich for ligation products in the 3C library that contain the interaction of interest and ‘trap’ the intervening fragments that were interacting simultaneously. After size selection to deplete PCR products <700 bp, the Nanopore MinION long-read sequencing platform is used to identify all trapped fragments. b, Overview of the number of trapped fragments per read detected in a C-Trap experiment in which the interaction between the R2 enhancer and the α-globin promoters was targeted in erythroid cells (two replicates). Read numbers represent reads in which both primer sequences could be detected at the ends. These numbers are much lower than for a Tri-C experiment (MEP_L_fig3Fig. 3b), owing to the relatively low output and read quality of the Nanopore MinION sequencing platform. The sensitivity of C-Trap is therefore ~100-fold lower than for Tri-C. Although C-Trap has the advantage of being capable of identifying many simultaneously interacting fragments in ligation products, the majority of the reads contain only a few trapped fragments. This could reflect the strength of the interaction between the R2 enhancer and the α-globin promoters, which are therefore often in close proximity in 3C ligation products, with few intervening fragments. It might also be related to a PCR amplification skew towards preferential enrichment of shorter fragments and lower quality of longer Nanopore reads. c, Comparison of multi-way interaction profiles generated from C-Trap reads containing interacting fragments with the R2 enhancer and the α-globin promoters, and from Tri-C reads containing both the R2 viewpoint and the α-globin promoters, in primary erythroid cells. The profiles look very similar: interactions are confined to the strongly compartmentalized ___domain and enriched over the enhancers. This confirms the validity of the multi-way interactions detected by Tri-C. Gene annotation (α-globin genes highlighted in red) and erythroid DNase I hypersensitivity (DHS) are shown at the top. The ___location of the C-Trap primers is indicated by green arrows. Coordinates (mm9): chr11:32,095,000–32,245,000.