High-throughput 3C-seq Analysis with r3Cseq

The 3C-seq experimental procedure is outlined in Figure 1, and the subsequent r3Cseq data analysis workflow is shown in Figure 2. Isolated cells are treated with a cross-linking agent to preserve in vivo nuclear proximity between DNA sequences. The DNA isolated from these cells is then digested using a primary restriction enzyme, typically a 6-bp cutting enzyme, such as HindIII, EcoRI or BamHI. The digested products are then ligated under diluted conditions to favor intra-molecular over inter-molecular ligation events. This digested and ligated chromatin yields composite sequences representing (distal) genomic regions that are in close physical proximity in the nuclear space. The digested and ligated chromatin is then de-cross-linked and subjected to a second restriction digest using a four-cutter (e.g. NlaIII or DpnII) as a secondary restriction enzyme to decrease the fragment sizes. The resulting digested DNA is then ligated again under diluted conditions, creating small circular fragments. These fragments are inverse PCR amplified using primers specific for a genomic region of interest (e.g. promoter, enhancer or any other element potentially involved in long-range interactions), termed the ‘viewpoint’. The amplified fragments are then sequenced using massively parallel high-throughput sequencing. The 3C-seq procedure produces DNA molecules consisting of viewpoint-specific primers followed by sequences derived from the ligated interacting fragments. These need to be trimmed in silico to remove the primer and viewpoint sequence, thus leaving only the captured sequence fragments for mapping (14 (link)). After trimming, reads are mapped against a reference genome using alignment software, such as Bowtie (18 (link)).
Figure 1.

3C-seq experimental procedures and data analysis workflow. Formaldehyde cross-linked chromatin is digested with a six-cutter restriction enzyme and ligated under dilute conditions. After de-cross-linking, DNA is digested with a four-cutter enzyme and again ligated under dilute conditions to create small circular fragments representing individual ligation events. Inverse PCR using viewpoint-specific primers containing Illumina sequencing adapters is used to generate a viewpoint-specific 3C-seq library. After high-throughput sequencing, reads are trimmed and mapped to the reference genome, after which they are loaded into the r3Cseq software.

Figure 2.

A summary of the r3Cseq analysis pipeline. The main features and the sequential order of operations are shown in the flow chart. In-depth discussion of the different operations and functions can be found in the ‘Materials and Methods’ and ‘Results’ sections.

Our r3Cseq package has been developed in the R statistical framework (19 ) as part of Bioconductor (20 (link)). It uses binary alignment/map (BAM)-aligned read files as input (21 (link)), which are generated by commonly used alignment software and carries out operations, such as class initialization, counting aligned reads per restriction fragment or per window size, read count normalization, statistical analysis of interactions in both cis and trans, data visualization and data export of the identified contacting regions. Figure 2 shows the main features and the sequential steps of the r3Cseq pipeline.