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Open Access Research article

Comparative analysis of 4C-Seq data generated from enzyme-based and sonication-based methods

Fan Gao12, Zong Wei1, Wange Lu1* and Kai Wang2*

Author Affiliations

1 Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Department of Biochemistry and Molecular Biology, University of Southern California, Los Angeles, CA 90089, USA

2 Zilkha Neurogenetic Institute, Department of Psychiatry and Department of Preventive Medicine, University of Southern California, Los Angeles, CA 90089, USA

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BMC Genomics 2013, 14:345  doi:10.1186/1471-2164-14-345

Published: 24 May 2013

Abstract

Background

Circular chromosome conformation capture, when coupled with next-generation sequencing (4C-Seq), can be used to identify genome-wide interaction of a given locus (a “bait” sequence) with all of its interacting partners. Conventional 4C approaches used restriction enzyme digestion to fragment chromatin, and recently sonication approach was also applied for this purpose. However, bioinformatics pipelines for analyzing sonication-based 4C-Seq data are not well developed. In addition, data consistency as well as similarity between the two methods has not been explored previously. Here we present a comparative analysis of 4C-Seq data generated by both methods, using an enhancer element of Pou5f1 gene in mouse embryonic stem (ES) cells.

Results

From biological replicates, we found good correlation (r>0.6) for inter-chromosomal interactions identified in either enzyme or sonication method. Compared to enzyme approach, sonication method generated less distal intra-chromosomal interactions, possibly due to the difference in chromatin fragmentation. From all mapped interactions, we further applied statistical models to identify enriched interacting regions. Interestingly, data generated from the two methods showed 30% overlap of the reproducible interacting regions. The interacting sites in the reproducible regions from both methods are similarly enriched with active histone marks. In addition, the interacting sites identified from sonication-based data are enriched with ChIP-Seq signals of transcription factors Oct4, Klf4, Esrrb, Tcfcp2i1, and Zfx that are critical for reprogramming and pluripotency.

Conclusions

Both enzyme-based and sonication-based 4C-Seq methods are valuable tools to explore long-range chromosomal interactions. Due to the nature of sonication-based method, correlation analysis of the 4C interactions with transcription factor binding should be more straightforward.

Keywords:
Circular chromosome conformation capture; Sonication; 4C-Seq; Bioinformatics; Next-generation sequencing