Here we demonstrated how to discover simultaneous multi-locus contacts using a straightforward conformation capture assay.
We next investigate whether the multi-locus contacts identified by the TM3C assay correspond to possible combinatorial regulatory interactions in KBM7 cells.
We identify multi-locus contacts involving three (triple) or four (quadruple) loci by a two-phase mapping strategy that separately maps chimeric subsequences within a single read.
We believe that with broader use of longer reads (e.g., Pacific Biosciences) TM3C will be able to profile a larger number of multi-locus contacts with higher signal-to-noise ratio.
Overall, we showed that TM3C provides a framework to identify multi-locus contacts genome-wide in conjunction with commonly used next generation sequencing platforms that produce short paired-end reads (e.g., 100 bp Illumina).
Despite these drawbacks, we believe that TM3C is an effective assay in profiling genome architecture evident by the consistency of our results with characteristic features of genome organization with the added benefit of revealing multi-locus contacts.
However, due to a number of differences in that protocol compared to TM3C (e.g., size selection for larger fragments, shorter read lengths and no in-gel ligation step), identifying a substantial number of multi-locus contacts was not possible when we apply our two-phase mapping pipeline to the Sexton et al. data (<0.0004% triples and no quadruples).
Focusing on a well-studied locus (IGF2-H19) thas has been shown to be involved in parent of origin specific long-range chromatin loops, we showed that TM3C identifies multi-locus contacts (triples), more than half of which were validated using PCR.
An earlier genome-wide assay by Sexton et al., which is adapted from the traditional Hi-C protocol and is similar to the assay we present here, acknowledged the existence of multi-locus contacts that can be identified from paired-end reads in their data [ 10].
Identifying multi-locus chromatin contacts in human cells using tethered multiple 3C.
To complement Tajima's D tests, a multi locus HKA test has been performed.
