Figure 2. Comparison of single and double oligonucleotide capture.

3C material generated from erythroid cells was captured using a single set of oligonucleotides designed to the α globin promoters (Supplementary Data). The two copies of the gene are virtually identical, therefore interaction profiles were generated from both genes simultaneously. After the first oligonucleotide capture step some of the material was sequenced using the Illumina MiSeq. The remaining library was used as input for a second round of oligonucleotide capture and then sequenced.

a. Comparison of the enrichment (to scale) resulting from the single (i) and double capture (iii) and the subsequent sequence read categorization following alignment (iii and iv). (i) Single capture resulted in 5-20,000 fold enrichment but only 0.3% of the reads contained a sequence that mapped to the captured fragment. (ii) Double capture increased the enrichment markedly; producing up to 3,000,000 fold enrichment. This dramatically increased the percentage of reads containing a restriction fragment that map to the capture region from 0.3% to 48.6%. The number of unique interactions was increased around 30-fold following double capture (from 10,832 to 327,787) (iii & iv) as library complexity now becomes the limiting factor.

b. Comparison of the raw informative interactions count per restriction enzyme fragment for single and double capture. The red vertical lines denote the location of captured viewpoints. The light blue lines highlight the five well described regulatory elements in the mouse (R1, R2, R3, R4 and HS-12). This showed that double capture did not notably alter the local interaction profile yet has 30-fold increased sensitivity.