title: Comparison of Hi-C results using in-solution versus in-nucleus ligation.
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.
authors: Nuclear Dynamics Programme, The Babraham Institute, Cambridge, CB22 3AT, UK. takashi.nagano@babraham.ac.uk.
authors: Takashi
authors: T
authors: Nagano
authors: Nuclear Dynamics Programme, The Babraham Institute, Cambridge, CB22 3AT, UK. csilla.varnai@babraham.ac.uk.
authors: Csilla
authors: C
authors: Várnai
authors: Nuclear Dynamics Programme, The Babraham Institute, Cambridge, CB22 3AT, UK. stefan.schoenfelder@babraham.ac.uk.
authors: Stefan
authors: S
authors: Schoenfelder
authors: Nuclear Dynamics Programme, The Babraham Institute, Cambridge, CB22 3AT, UK. biola-maria.javierre@babraham.ac.uk.
authors: Biola-Maria
authors: BM
authors: Javierre
authors: Nuclear Dynamics Programme, The Babraham Institute, Cambridge, CB22 3AT, UK. steven.wingett@babraham.ac.uk.
authors: Steven W
authors: SW
authors: Wingett
authors: Nuclear Dynamics Programme, The Babraham Institute, Cambridge, CB22 3AT, UK. peter.fraser@babraham.ac.uk.
authors: Peter
authors: P
authors: Fraser
mesh_terms: D000818:Animals; D002467:Cell Nucleus / Q000235:genetics; D002478:Cells, Cultured; D002843:Chromatin / Q000737:chemistry*; D023281:Genomics / Q000379:methods*; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL
chemical_list: D002843:Chromatin