Moving Away from Proximal Ligation to Study Higher Order Chromatin Complexes at High Resolution
Abstract: Genome organization is increasingly believed to influence and participate in gene regulation. Thestudy of the interactions that constitute this organization is increasingly performed using chromosomeconformation capture (3C) technologies. While good for the study of pairwise interactions betweenelements, higher order structures are inaccessible due to the use of proximal ligation to link elements.This thesis thus endeavored to develop a method to study these higher order structures in an effort tomove away from proximal ligation. Instead of pairwise linking interacting fragments through ligation,strategies for barcoding of chromatin complexes were tested. These complexes were extracted fromcrosslinked cells and captured using chromatin immunoprecipitation (ChIP) procedure. The fragmentswithin the complex were then fitted with adaptors and barcodes according to two approaches. The first(called Approach A) being adaptor ligation with subsequent ligation onto barcoded beads, the second(called Approach B) tagmentation with subsequent barcoding through emulsion PCR (emPCR).Several steps of optimization were performed and a total six libraries created and sequenced, twousing approach A and four using approach B. Analysis of these libraries demonstrated progress inseveral key areas such as barcode clusters containing multiple fragments and phasing. In the B datasetswas also found an enrichment form short range interactions, in accordance with 3C observations. TheB datasets outperformed A in most regards and was thus deemed the preferred path for future studies.The greatest challenge yet to overcome is to lower duplication rates which currently are at a minimumof 79%. To decrease rates several parameters for optimization have been identified for futuredevelopment.
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