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

BackgroundChromosome 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.ResultsWe 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.ConclusionsWe 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.

[1]  Job Dekker,et al.  The three 'C' s of chromosome conformation capture: controls, controls, controls , 2005, Nature Methods.

[2]  I. Amit,et al.  Comprehensive mapping of long range interactions reveals folding principles of the human genome , 2011 .

[3]  C. Glass,et al.  Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. , 2010, Molecular cell.

[4]  T. Fennell,et al.  Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries , 2011, Genome Biology.

[5]  A. Tanay,et al.  Probabilistic modeling of Hi-C contact maps eliminates systematic biases to characterize global chromosomal architecture , 2011, Nature Genetics.

[6]  Giacomo Cavalli,et al.  A chromatin insulator driving three-dimensional Polycomb response element (PRE) contacts and Polycomb association with the chromatin fiber , 2011, Proceedings of the National Academy of Sciences.

[7]  A. Tanay,et al.  Three-Dimensional Folding and Functional Organization Principles of the Drosophila Genome , 2012, Cell.

[8]  Reza Kalhor,et al.  Genome architectures revealed by tethered chromosome conformation capture and population-based modeling , 2011, Nature Biotechnology.

[9]  Data production leads,et al.  An integrated encyclopedia of DNA elements in the human genome , 2012 .

[10]  J. Sedat,et al.  Spatial partitioning of the regulatory landscape of the X-inactivation centre , 2012, Nature.

[11]  ENCODEConsortium,et al.  An Integrated Encyclopedia of DNA Elements in the Human Genome , 2012, Nature.

[12]  Lee E. Edsall,et al.  A map of the cis-regulatory sequences in the mouse genome , 2012, Nature.

[13]  Jesse R. Dixon,et al.  Topological Domains in Mammalian Genomes Identified by Analysis of Chromatin Interactions , 2012, Nature.

[14]  L. Mirny,et al.  Iterative Correction of Hi-C Data Reveals Hallmarks of Chromosome Organization , 2012, Nature Methods.

[15]  Tom Misteli,et al.  SnapShot: Chromosome Conformation Capture , 2012, Cell.

[16]  Michael S. Becker,et al.  Spatial Organization of the Mouse Genome and Its Role in Recurrent Chromosomal Translocations , 2012, Cell.

[17]  L. Mirny,et al.  Exploring the three-dimensional organization of genomes: interpreting chromatin interaction data , 2013, Nature Reviews Genetics.

[18]  Bing Ren,et al.  Whole-genome haplotype reconstruction using proximity-ligation and shotgun sequencing , 2013, Nature Biotechnology.

[19]  A. Tanay,et al.  Single cell Hi-C reveals cell-to-cell variability in chromosome structure , 2013, Nature.

[20]  Sergey V. Razin,et al.  Disclosure of a structural milieu for the proximity ligation reveals the elusive nature of an active chromatin hub , 2013, Nucleic acids research.

[21]  G. Schroth,et al.  Cohesin-mediated interactions organize chromosomal domain architecture , 2013, The EMBO journal.

[22]  A. Ashworth,et al.  Unbiased analysis of potential targets of breast cancer susceptibility loci by Capture Hi-C , 2014, Genome research.

[23]  Peter H. L. Krijger,et al.  Targeted sequencing by proximity ligation for comprehensive variant detection and local haplotyping , 2014, Nature Biotechnology.

[24]  Neva C. Durand,et al.  A 3D Map of the Human Genome at Kilobase Resolution Reveals Principles of Chromatin Looping , 2014, Cell.

[25]  D. Odom,et al.  Comparative Hi-C Reveals that CTCF Underlies Evolution of Chromosomal Domain Architecture , 2015, Cell reports.

[26]  R. Houlston,et al.  Capture Hi-C identifies the chromatin interactome of colorectal cancer risk loci , 2015, Nature Communications.

[27]  Cameron S. Osborne,et al.  The pluripotent regulatory circuitry connecting promoters to their long-range interacting elements , 2015, Genome research.