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Genome Biol. 2015 Aug 26;16:175. doi: 10.1186/s13059-015-0753-7.

Comparison of Hi-C results using in-solution versus in-nucleus ligation.

Author information

1
Nuclear Dynamics Programme, The Babraham Institute, Cambridge, CB22 3AT, UK. takashi.nagano@babraham.ac.uk.
2
Nuclear Dynamics Programme, The Babraham Institute, Cambridge, CB22 3AT, UK. csilla.varnai@babraham.ac.uk.
3
Nuclear Dynamics Programme, The Babraham Institute, Cambridge, CB22 3AT, UK. stefan.schoenfelder@babraham.ac.uk.
4
Nuclear Dynamics Programme, The Babraham Institute, Cambridge, CB22 3AT, UK. biola-maria.javierre@babraham.ac.uk.
5
Nuclear Dynamics Programme, The Babraham Institute, Cambridge, CB22 3AT, UK. steven.wingett@babraham.ac.uk.
6
Nuclear Dynamics Programme, The Babraham Institute, Cambridge, CB22 3AT, UK. peter.fraser@babraham.ac.uk.

Abstract

BACKGROUND:

Chromosome conformation capture and various derivative methods such as 4C, 5C and Hi-C have emerged as standard tools to analyze the three-dimensional organization of the genome in the nucleus. These methods employ ligation of diluted cross-linked chromatin complexes, intended to favor proximity-dependent, intra-complex ligation. During development of single-cell Hi-C, we devised an alternative Hi-C protocol with ligation in preserved nuclei rather than in solution. Here we directly compare Hi-C methods employing in-nucleus ligation with the standard in-solution ligation.

RESULTS:

We show in-nucleus ligation results in consistently lower levels of inter-chromosomal contacts. Through chromatin mixing experiments we show that a significantly large fraction of inter-chromosomal contacts are the result of spurious ligation events formed during in-solution ligation. In-nucleus ligation significantly reduces this source of experimental noise, and results in improved reproducibility between replicates. We also find that in-nucleus ligation eliminates restriction fragment length bias found with in-solution ligation. These improvements result in greater reproducibility of long-range intra-chromosomal and inter-chromosomal contacts, as well as enhanced detection of structural features such as topologically associated domain boundaries.

CONCLUSIONS:

We conclude that in-nucleus ligation captures chromatin interactions more consistently over a wider range of distances, and significantly reduces both experimental noise and bias. In-nucleus ligation creates higher quality Hi-C libraries while simplifying the experimental procedure. We suggest that the entire range of 3C applications are likely to show similar benefits from in-nucleus ligation.

PMID:
26306623
PMCID:
PMC4580221
DOI:
10.1186/s13059-015-0753-7
[Indexed for MEDLINE]
Free PMC Article

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