BACKGROUND
the spatial organization of the eukaryotic genome is now accessible through techniques involving massive parallel high-throughput sequencing .
an understanding of how chromosomes fold can provide insight into complex relationships between chromatin structure, genetic activity and functional state of the cell .
in addition, genome wide chromatin interaction data sets can reveal long-range information about the grouping and linear organization of sequences along entire chromosomes, enabling high quality chromosome-scale de novo genome assembly  <cit> .
chromosome conformation capture -based techniques have emerged as powerful tools for mapping chromatin contacts .
one recently-described technique, tethered conformation capture   <cit> , was developed to improve signal to noise ratio over previously published techniques, allowing in-depth analysis of both intra and inter-chromosomal contacts.
3c-based techniques use proximity ligation and massively parallel sequencing to probe the three-dimensional architecture of chromosomes within the nucleus, with closely interacting regions captured via the ligation step and identified through sequence analysis.
in the resulting data sets, the probability of intra-chromosomal contacts is on average much higher than that of inter-chromosomal contacts, as expected if chromosomal territories are at least partially distinct.
these data have supported a model in which chromosomal territories are indeed distinct entities: although the probability of interaction decays with linear distance, even loci separated by megabases on the same chromosome are more likely to interact than loci on different chromosomes.
we have developed a rapid tethered conformation capture  technique to allow fast application of a tcc-based protocol in c. elegans.
our method allows detection of chromatin contacts using unfractionated whole tissues or whole organisms as starting material, avoiding extensive cellular and molecular fractionation steps.
applied to c. elegans, this protocol illuminates both the large scale structural partitioning  and fine resolution genome architecture dynamics .
RESULTS
detecting genome-wide chromatin contacts using rtcc
rtcc differs from previous chromosome conformation capture protocols in two key respects:  the lack of a need for cellular or chromatin fractionation,  the application of efficient transposon tagging approach toward capture of potential ligation junctions.
figure 1b outlines the rtcc protocol with approximations of time for each step.
of note are the avoidance of cellular or chromatin fractionation steps in standard hi-c, and the adaptation of the protocol for low input volumes in sequencing library preparation by adapting nextera tagmentase  <cit>  to sequencing of biotin-labeled junctions.fig.
<dig> overview of rtcc protocol.
a diagram shows a schematic description of steps from a crude tissue homogenate to a proximity sequencing library .
for our studies , animals flash-frozen in liquid nitrogen were finely ground using either mortar and pestle or using an electric drill with “cellcrusher” drill-bit and “cellcrusher” base held at liquid nitrogen temperature and treated with formaldehyde to covalently cross-link proteins to each other and to dna .
chromatin is solubilized with detergent and proteins were non-specifically biotinylated .
dna was digested with a restriction enzyme that generates 5’ overhangs.
cross-linked complexes were immobilized at a very low density on the surface of streptavidin-coated magnetic beads  through the biotinylated proteins, while the non-cross-linked dna fragments were removed.
5′ overhangs were filled in using dna polymerase and a nucleotide mixture containing biotin-14-dctp  to generate blunt ends.
blunt dna ends were ligated.
cross-linking was reversed and dna was purified.
the dna was fragmented and tagged  using nextera tagmentase.
dna fragments containing biotinylated ctp were selected on streptavidin-coated beads.
this selects for ligation junctions and dna molecules biotinylated at their terminus.
a sequencing library was generated via pcr using the nextera  adaptors introduced at step  <dig>  this amplification step should provide a substantial enrichment for ligation junctions, since molecules that were biotinylated solely on their termini would carry a nextera adaptor only on one side.
b rtcc protocol timeline
to preserve native features of genomic organization, whole worms were flash frozen and finely ground under liquid nitrogen.
these frozen samples were quickly resuspended and subjected to formaldehyde treatment to chemically crosslink dna and proteins.
this material could then be used directly for molecular manipulations and analysis , avoiding any need for an intervening nuclear or chromatin isolation step.
in the cross-linked lysates, dna was digested with a restriction enzyme, proteins were biotinylated non-specifically   <cit> , protein-dna cross-linked complexes were immobilized at a low surface density on streptavidin-coated beads, and physically juxtaposed free dna ends were filled in with a nucleotide mixture containing biotin-14-dctp.
ligation of ends was then performed while fragments remained tethered to the surface of the beads.
following reversal of crosslinking and release of dna, the nextera tagmentation  protocol was used to fragment the dna and add linkers for sequencing in a single step  <cit> .
the ligation junctions were purified by selection for biotinylated dna fragments, which were subjected to massively parallel sequencing.
mapping of the sequenced junctions allowed detection of genomic locations for pairs of contacting loci.
we have applied rtcc to a variety of different tissue samples derived from c. elegans, using the dpnii, avaii, and hindiii restriction enzymes, which cut respectively with  <dig>   <dig> , and  <dig> base recognition specificities  exemplary chromatin interaction intensity maps are shown in fig.
<dig>  we then applied several approaches in evaluating the consistency and accuracy of this large-scale dataset.fig.
<dig> chromatin interaction intensity maps.
a heat map showing raw counts of observed chromatin contacts on a genome-wide scale with 50kb bins .
b binned chromatin interaction map for wild type n <dig> young adults displayed with color representing the log <dig> of the observed/expected ratio for each 50kb bin pair.
c magnified log <dig> plot as in b, but focused just on chromosome i. d a further normalization of the plot in panel c in which the interaction level for each combination of 50kb intervals is normalized to other pairs of intervals separated by a similar distance .
e log <dig> of the observed/expected ratio of interaction frequency  for the x chromosome.
f coverage and distance normalized interaction plot  for the x chromosome
first, we evaluated the consistency of results from this approach by comparing results from different experimental replicates.
the replicates were carried out with slight deviations in the underlying protocol, distinct stages and tissue distributions, and different choices of restriction enzymes; hence their consistency becomes a test for both biological and technical reproducibility.
indeed we observed that experimental replicates  were highly correlated .
second we compared the chromatin interaction matrices obtained with rtcc with a recent hi-c analysis performed using c. elegans embryos, acquired by crane et al.
<cit>  towards understanding of x chromosome topology remodeling during dosage compensation.
this comparison likewise demonstrates experiment-to-experiment correlation, both by inspection  and from calculating a formal pearson correlation coefficient .
further validation of the rtcc data that we obtained comes from analysis of distances between interacting regions captured by rtcc ligation.
with any proximity ligation protocol , we expect a subset of artefactual ligation events that will join dna segments with no true association .
such events might be expected to lack a clear dependence on inter-locus distance, while bone-fide contacts that were captured from a physiological chromosome configuration would be expected to be much more frequent for closely linked loci.
our data shows an expected and dramatic inverse association between the distance between the interacting loci and the number of intra-chromosomal contacts .
dependence of the chromatin contacts on the distance between interacting regions is consistent with dna polymer-like behavior in which three-dimensional distance between loci increases with increasing genomic distance  <cit> .
analysis of frequencies of different types of contacts shows greater frequency of intra-chromosomal contacts than inter-chromosomal  supporting the idea of chromosome territoriality  <cit> .fig.
<dig> interaction frequency decay as function of the distance between interacting loci.
the genome was divided into 1kb non-overlapping intervals and intra-chromosomal contacts between these intervals were counted to produce the plotted profile.
in the chart we are plotting the fraction of the total intra-chromosomal junctions detected within  <dig> bp of a given genomic distance
fig.
<dig> differences in prevalence of intra- and inter-chromosomal contacts.
contacts detected in aggregated n <dig> dpnii experiments  were used to construct an interaction frequency matrix with resolution of 50kb.
a, b, c show sections of this chart under different magnifications.
for each square in the matrix we calculated an expected number of contacts  and compared these with the quantity of the observed “junctional” reads between the two indicated regions.
we have plotted the frequencies of observed/expected ratios in intra- and inter-chromosomal junctions for each bin of width  <dig>  in observed/expected value.
junctions mapping to ribosomal rna sequence on chromosome i were excluded from the calculation
chromatin interaction maps
using the interaction information we obtained from the mapping of the sequenced dna library , we created a genome-wide raw chromatin interaction matrix.
in order to create this matrix, the genome was divided into segments, the size of which depends on the depth of sequencing analysis and level of resolution required .
each cell in the matrix mi,j corresponds to the number of contacts  between segment i and segment j of the genome.
the interaction matrix can be depicted visually with a heat map, in which the color intensity correlates with contact frequency .
a whole genome raw contacts map for c. elegans shows  <dig> distinct squares aligned on the diagonal, each corresponding to an individual chromosome, supporting the idea that the probability of intra-chromosomal contacts is on average much higher than that of inter-chromosomal contacts and that chromosomes occupy distinct territories.
the whole genome chromatin interaction matrix was normalized using the expected number of contacts assuming each region has an equal chance of interacting with every other region in the genome, essentially normalizing to read coverage at each region.
the resulting normalized whole genome matrix, presented as a heat map in fig.
2b, exhibits more clear separation into  <dig> chromosomes and shows some of the intra-chromosomal organization features as well.
zooming in to the single chromosome level  allows study of intra-chromosomal structural organization features .
tendencies for arm-arm and center-center interaction on the autosomes are evident from two dimensional heat maps that associate each combination of genome positions  with a color indicating the degree of over- or under-representation in the population of novel junctions.
this observation is indicative of a physical basis for the observation that c. elegans autosomes show distinct features in arm and center regions .
these findings are consistent with results from linear analyses of chromosomal features , from cytological studies  <cit>  and from another recently communicated chromosome capture analysis by crane et al.
<cit> .
additional file 3: figure s <dig> shows a remarkable association between center-arm positioning within each autosome for our data and data from crane et al.
<cit> .
one physical correlate of the association is suggested by alignment with a dataset derived from immunoprecipitation  <cit>  using an antibody against the nuclear envelope component lem- <dig> .
as noted also by crane et al.
<cit>  our data point to a more complex organization on the x chromosome, with evidence for a domain organization that differs from the end-center-end organization observed on the five autosomes.
several years ago, it was noted that a subset of dna segments on c. elegans autosomal arms exhibit a strongly periodic sequence character, with phased runs of a and t residues.
known as the “patcs” regions, these genomic features are characteristic of introns and other noncoding sequences for a subset of genes expressed in the c. elegans germ line  <cit> .
we found that patc character  was strongly associated with partitioning of contacts between central and arm regions of chromosomes  this tendency appears to be a general property of this unusual genomic partition, as all five autosomes show similar arm-association-with-arm enrichment on both arms .
this correspondence suggests a strong tendency for patc-rich regions to inhabit their own subdomain of the connectivity network, and is consistent with proposed roles of such extended dna sequence features in long range chromosomal organization  <cit> .table  <dig> correlation between phased an/tn cluster  content and preference for interaction with chromosome arms
to measure correlation of inter-chromosomal contacts between chromosomal arms regions  and patc enrichment we divided the genome into 25kb  non-overlapping intervals.
for each interval we measured the enrichment for patc by counting the number of locations with high patc score .
in order to measure the level of enrichment of inter-chromosomal arm-arm contacts over the arm-centers contacts we calculate the number of inter-chromosomal arm-arm contacts and number of inter-chromosomal arm-center contacts for every interval.
log <dig> value of the enrichment for inter-chromosomal arm-arm contacts is used to calculate correlation with patc enrichment in different genomic regions
modeling-based evaluation suggests utility of rtcc in identification of chromosome structural variation
information on genomic architecture from hi-c and similar approaches has been useful in diverse genome-structure applications .
to evaluate the utility of rtcc in investigating structural variation, we produced three conceptual “model reference genomes” with substantial structural variation, one where the standard n <dig> reference genome is related by a chromosomal fusion, one where the n <dig> genome is related by a reciprocal translocation, and one where the n <dig> genome is related by a large inversion.
to model “real world” structural variants, we constructed the translocation with breakpoints at a homologous point in an extended dna repeat .
this rearrangement would have remained undetected with paired end or split read sequencing.
as shown in fig.
<dig> all of the simulated structural variants could be readily detected using rtcc analysis.fig.
<dig> detection of simulated structural variants by rtcc.
this figure shows an analytical evaluation of feasibility for using rtcc data to detect large scale structural variations in the c. elegans genome.
for this analysis, the n <dig> reference genome was computationally modified to simulate “model reference genomes” that differ structurally from the normal c. elegans genome.
we then analyzed the data from n <dig>  experiments as in fig.
<dig>  aligning the reads to the indicated simulated reference genome and using a 50kb window size as above.
in each case, the analysis yields a footprint characteristic of the simulated rearrangement.
in , we simulated a two-chromosome fusion: we generated a model reference genome in which chri was artificially separated into  <dig> parts , each  <dig> mb long.
execution of our analysis pipeline resulted in visible evidence for a high level of contacts between the artificially created right tip of i-l and the artificially created left tip of i-r of the model reference genome, which are “fused” in the n <dig> genome.
in , we simulated a reciprocal translocation by creating a model reference genome in which segments of chromosomes ii and iv were virtually recombined.
the simulated recombination was created in the middle of a tc <dig> transposable element  present in multiple copies in the genome to simulate a rearrangement that would have presented detection challenges by standard methods.
the “translocation” in the n <dig> data  is evident on the plot, by the distinct accentuation of contacts between ii-l and ii-r and iv-l and iv-r. in , we generated a model reference genome in which a large inversion  was virtually introduced on chromosome i. evidence for inversion is visible on the plot
CONCLUSIONS
in this study we modified and optimized tcc protocol to work with limited amounts of unfractionated tissue, avoiding subcellular fractionation and chromatin isolation.
the ability to measure proximity between genomic loci in linear space can be utilized both in structural studies of chromosomes and in applications such as genome assembly or phasing.
the rtcc protocol that we present here should provide an efficient and robust means of performing hi-c experiments in general and in nematodes in particular.
our method allows detection of long-range contacts between genomic loci, supplying useful information to study three-dimensional organization of the genome.
further, hi-c data sets generated using our rtcc protocol can provide a measurement of proximity between genomic elements in linear sense, allowing this knowledge to be used in bioinformatics applications such as lachesis for high quality genome assembly  <cit> .

 the spatial organization of the eukaryotic genome is now accessible through techniques involving massive parallel high-throughput sequencing. an understanding of how chromosomes fold can provide insight into complex relationships between chromatin structure, genetic activity and functional state of the cell. in addition, genome wide chromatin interaction data sets can reveal long-range information about the grouping and linear organization of sequences along entire chromosomes, enabling high quality chromosome-scale de novo genome assembly. chromosome conformation capture -based techniques have emerged as powerful tools for mapping chromatin contacts. one recently-described technique, tethered conformation capture, was developed to improve signal to noise ratio over previously published techniques, allowing in-depth analysis of both intra and inter-chromosomal contacts. 3c-based techniques use proximity ligation and massively parallel sequencing to probe the three-dimensional architecture of chromosomes within the nucleus, with closely interacting regions captured via the ligation step and identified through sequence analysis. in the resulting data sets, the probability of intra-chromosomal contacts is on average much higher than that of inter-chromosomal contacts, as expected if chromosomal territories are at least partially distinct. these data have supported a model in which chromosomal territories are indeed distinct entities: although the probability of interaction decays with linear distance, even loci separated by megabases on the same chromosome are more likely to interact than loci on different chromosomes. we have developed a rapid tethered conformation capture technique to allow fast application of a tcc-based protocol in c. elegans. our method allows detection of chromatin contacts using unfractionated whole tissues or whole organisms as starting material, avoiding extensive cellular and molecular fractionation steps. applied to c. elegans, this protocol illuminates both the large scale structural partitioning and fine resolution genome architecture dynamics.  detecting genome-wide chromatin contacts using rtcc rtcc differs from previous chromosome conformation capture protocols in two key respects: the lack of a need for cellular or chromatin fractionation, the application of efficient transposon tagging approach toward capture of potential ligation junctions. figure 1b outlines the rtcc protocol with approximations of time for each step. of note are the avoidance of cellular or chromatin fractionation steps in standard hi-c, and the adaptation of the protocol for low input volumes in sequencing library preparation by adapting nextera tagmentase to sequencing of biotin-labeled junctions.fig. overview of rtcc protocol. a diagram shows a schematic description of steps from a crude tissue homogenate to a proximity sequencing library. for our studies, animals flash-frozen in liquid nitrogen were finely ground using either mortar and pestle or using an electric drill with “cellcrusher” drill-bit and “cellcrusher” base held at liquid nitrogen temperature and treated with formaldehyde to covalently cross-link proteins to each other and to dna. chromatin is solubilized with detergent and proteins were non-specifically biotinylated. dna was digested with a restriction enzyme that generates 5’ overhangs. cross-linked complexes were immobilized at a very low density on the surface of streptavidin-coated magnetic beads through the biotinylated proteins, while the non-cross-linked dna fragments were removed. 5′ overhangs were filled in using dna polymerase and a nucleotide mixture containing biotin-14-dctp to generate blunt ends. blunt dna ends were ligated. cross-linking was reversed and dna was purified. the dna was fragmented and tagged using nextera tagmentase. dna fragments containing biotinylated ctp were selected on streptavidin-coated beads. this selects for ligation junctions and dna molecules biotinylated at their terminus. a sequencing library was generated via pcr using the nextera adaptors introduced at step this amplification step should provide a substantial enrichment for ligation junctions, since molecules that were biotinylated solely on their termini would carry a nextera adaptor only on one side. b rtcc protocol timeline to preserve native features of genomic organization, whole worms were flash frozen and finely ground under liquid nitrogen. these frozen samples were quickly resuspended and subjected to formaldehyde treatment to chemically crosslink dna and proteins. this material could then be used directly for molecular manipulations and analysis, avoiding any need for an intervening nuclear or chromatin isolation step. in the cross-linked lysates, dna was digested with a restriction enzyme, proteins were biotinylated non-specifically, protein-dna cross-linked complexes were immobilized at a low surface density on streptavidin-coated beads, and physically juxtaposed free dna ends were filled in with a nucleotide mixture containing biotin-14-dctp. ligation of ends was then performed while fragments remained tethered to the surface of the beads. following reversal of crosslinking and release of dna, the nextera tagmentation protocol was used to fragment the dna and add linkers for sequencing in a single step. the ligation junctions were purified by selection for biotinylated dna fragments, which were subjected to massively parallel sequencing. mapping of the sequenced junctions allowed detection of genomic locations for pairs of contacting loci. we have applied rtcc to a variety of different tissue samples derived from c. elegans, using the dpnii, avaii, and hindiii restriction enzymes, which cut respectively with, and base recognition specificities exemplary chromatin interaction intensity maps are shown in fig.  we then applied several approaches in evaluating the consistency and accuracy of this large-scale dataset.fig. chromatin interaction intensity maps. a heat map showing raw counts of observed chromatin contacts on a genome-wide scale with 50kb bins. b binned chromatin interaction map for wild type n young adults displayed with color representing the log of the observed/expected ratio for each 50kb bin pair. c magnified log plot as in b, but focused just on chromosome i. d a further normalization of the plot in panel c in which the interaction level for each combination of 50kb intervals is normalized to other pairs of intervals separated by a similar distance. e log of the observed/expected ratio of interaction frequency for the x chromosome. f coverage and distance normalized interaction plot for the x chromosome first, we evaluated the consistency of results from this approach by comparing results from different experimental replicates. the replicates were carried out with slight deviations in the underlying protocol, distinct stages and tissue distributions, and different choices of restriction enzymes; hence their consistency becomes a test for both biological and technical reproducibility. indeed we observed that experimental replicates were highly correlated. second we compared the chromatin interaction matrices obtained with rtcc with a recent hi-c analysis performed using c. elegans embryos, acquired by crane et al. towards understanding of x chromosome topology remodeling during dosage compensation. this comparison likewise demonstrates experiment-to-experiment correlation, both by inspection and from calculating a formal pearson correlation coefficient. further validation of the rtcc data that we obtained comes from analysis of distances between interacting regions captured by rtcc ligation. with any proximity ligation protocol, we expect a subset of artefactual ligation events that will join dna segments with no true association. such events might be expected to lack a clear dependence on inter-locus distance, while bone-fide contacts that were captured from a physiological chromosome configuration would be expected to be much more frequent for closely linked loci. our data shows an expected and dramatic inverse association between the distance between the interacting loci and the number of intra-chromosomal contacts. dependence of the chromatin contacts on the distance between interacting regions is consistent with dna polymer-like behavior in which three-dimensional distance between loci increases with increasing genomic distance. analysis of frequencies of different types of contacts shows greater frequency of intra-chromosomal contacts than inter-chromosomal supporting the idea of chromosome territoriality.fig. interaction frequency decay as function of the distance between interacting loci. the genome was divided into 1kb non-overlapping intervals and intra-chromosomal contacts between these intervals were counted to produce the plotted profile. in the chart we are plotting the fraction of the total intra-chromosomal junctions detected within bp of a given genomic distance fig. differences in prevalence of intra- and inter-chromosomal contacts. contacts detected in aggregated n dpnii experiments were used to construct an interaction frequency matrix with resolution of 50kb. a, b, c show sections of this chart under different magnifications. for each square in the matrix we calculated an expected number of contacts and compared these with the quantity of the observed “junctional” reads between the two indicated regions. we have plotted the frequencies of observed/expected ratios in intra- and inter-chromosomal junctions for each bin of width in observed/expected value. junctions mapping to ribosomal rna sequence on chromosome i were excluded from the calculation chromatin interaction maps using the interaction information we obtained from the mapping of the sequenced dna library, we created a genome-wide raw chromatin interaction matrix. in order to create this matrix, the genome was divided into segments, the size of which depends on the depth of sequencing analysis and level of resolution required. each cell in the matrix mi,j corresponds to the number of contacts between segment i and segment j of the genome. the interaction matrix can be depicted visually with a heat map, in which the color intensity correlates with contact frequency. a whole genome raw contacts map for c. elegans shows distinct squares aligned on the diagonal, each corresponding to an individual chromosome, supporting the idea that the probability of intra-chromosomal contacts is on average much higher than that of inter-chromosomal contacts and that chromosomes occupy distinct territories. the whole genome chromatin interaction matrix was normalized using the expected number of contacts assuming each region has an equal chance of interacting with every other region in the genome, essentially normalizing to read coverage at each region. the resulting normalized whole genome matrix, presented as a heat map in fig. 2b, exhibits more clear separation into chromosomes and shows some of the intra-chromosomal organization features as well. zooming in to the single chromosome level allows study of intra-chromosomal structural organization features. tendencies for arm-arm and center-center interaction on the autosomes are evident from two dimensional heat maps that associate each combination of genome positions with a color indicating the degree of over- or under-representation in the population of novel junctions. this observation is indicative of a physical basis for the observation that c. elegans autosomes show distinct features in arm and center regions. these findings are consistent with results from linear analyses of chromosomal features, from cytological studies and from another recently communicated chromosome capture analysis by crane et al.. additional file 3: figure s shows a remarkable association between center-arm positioning within each autosome for our data and data from crane et al.. one physical correlate of the association is suggested by alignment with a dataset derived from immunoprecipitation using an antibody against the nuclear envelope component lem-. as noted also by crane et al. our data point to a more complex organization on the x chromosome, with evidence for a domain organization that differs from the end-center-end organization observed on the five autosomes. several years ago, it was noted that a subset of dna segments on c. elegans autosomal arms exhibit a strongly periodic sequence character, with phased runs of a and t residues. known as the “patcs” regions, these genomic features are characteristic of introns and other noncoding sequences for a subset of genes expressed in the c. elegans germ line. we found that patc character was strongly associated with partitioning of contacts between central and arm regions of chromosomes this tendency appears to be a general property of this unusual genomic partition, as all five autosomes show similar arm-association-with-arm enrichment on both arms. this correspondence suggests a strong tendency for patc-rich regions to inhabit their own subdomain of the connectivity network, and is consistent with proposed roles of such extended dna sequence features in long range chromosomal organization.table correlation between phased an/tn cluster content and preference for interaction with chromosome arms to measure correlation of inter-chromosomal contacts between chromosomal arms regions and patc enrichment we divided the genome into 25kb non-overlapping intervals. for each interval we measured the enrichment for patc by counting the number of locations with high patc score. in order to measure the level of enrichment of inter-chromosomal arm-arm contacts over the arm-centers contacts we calculate the number of inter-chromosomal arm-arm contacts and number of inter-chromosomal arm-center contacts for every interval. log value of the enrichment for inter-chromosomal arm-arm contacts is used to calculate correlation with patc enrichment in different genomic regions modeling-based evaluation suggests utility of rtcc in identification of chromosome structural variation information on genomic architecture from hi-c and similar approaches has been useful in diverse genome-structure applications. to evaluate the utility of rtcc in investigating structural variation, we produced three conceptual “model reference genomes” with substantial structural variation, one where the standard n reference genome is related by a chromosomal fusion, one where the n genome is related by a reciprocal translocation, and one where the n genome is related by a large inversion. to model “real world” structural variants, we constructed the translocation with breakpoints at a homologous point in an extended dna repeat. this rearrangement would have remained undetected with paired end or split read sequencing. as shown in fig.  all of the simulated structural variants could be readily detected using rtcc analysis.fig. detection of simulated structural variants by rtcc. this figure shows an analytical evaluation of feasibility for using rtcc data to detect large scale structural variations in the c. elegans genome. for this analysis, the n reference genome was computationally modified to simulate “model reference genomes” that differ structurally from the normal c. elegans genome. we then analyzed the data from n experiments as in fig.  aligning the reads to the indicated simulated reference genome and using a 50kb window size as above. in each case, the analysis yields a footprint characteristic of the simulated rearrangement. in, we simulated a two-chromosome fusion: we generated a model reference genome in which chri was artificially separated into parts, each mb long. execution of our analysis pipeline resulted in visible evidence for a high level of contacts between the artificially created right tip of i-l and the artificially created left tip of i-r of the model reference genome, which are “fused” in the n genome. in, we simulated a reciprocal translocation by creating a model reference genome in which segments of chromosomes ii and iv were virtually recombined. the simulated recombination was created in the middle of a tc transposable element present in multiple copies in the genome to simulate a rearrangement that would have presented detection challenges by standard methods. the “translocation” in the n data is evident on the plot, by the distinct accentuation of contacts between ii-l and ii-r and iv-l and iv-r. in, we generated a model reference genome in which a large inversion was virtually introduced on chromosome i. evidence for inversion is visible on the plot  in this study we modified and optimized tcc protocol to work with limited amounts of unfractionated tissue, avoiding subcellular fractionation and chromatin isolation. the ability to measure proximity between genomic loci in linear space can be utilized both in structural studies of chromosomes and in applications such as genome assembly or phasing. the rtcc protocol that we present here should provide an efficient and robust means of performing hi-c experiments in general and in nematodes in particular. our method allows detection of long-range contacts between genomic loci, supplying useful information to study three-dimensional organization of the genome. further, hi-c data sets generated using our rtcc protocol can provide a measurement of proximity between genomic elements in linear sense, allowing this knowledge to be used in bioinformatics applications such as lachesis for high quality genome assembly. 
our streamlined protocol provides an accelerated, robust, and broadly applicable means of generating chromatin spatial organization maps and detecting genome rearrangements without a need for cellular or chromatin fractionation. we applied the method to caenorhabditis elegans, obtaining chromatin interaction maps that provide a sequence-anchored delineation of salient aspects of caenorhabditis elegans chromosome structure, demonstrating a high level of consistency in overall chromosome organization between biological samples collected under different conditions.
 identification of locus-locus contacts at the chromatin level provides a valuable foundation for understanding of nuclear architecture and function and a valuable tool for inferring long-range linkage relationships. as one approach to this, chromatin conformation capture-based techniques allow creation of genome spatial organization maps. while such approaches have been available for some time, methodological advances will be of considerable use in minimizing both time and input material required for successful application.  here we report a modified tethered conformation capture protocol that utilizes a series of rapid and efficient molecular manipulations. we applied the method to caenorhabditis elegans, obtaining chromatin interaction maps that provide a sequence-anchored delineation of salient aspects of caenorhabditis elegans chromosome structure, demonstrating a high level of consistency in overall chromosome organization between biological samples collected under different conditions. in addition to the application of the method to defining nuclear architecture, we found the resulting chromatin interaction maps to be of sufficient resolution and sensitivity to enable detection of large-scale structural variants such as inversions or translocations.  our streamlined protocol provides an accelerated, robust, and broadly applicable means of generating chromatin spatial organization maps and detecting genome rearrangements without a need for cellular or chromatin fractionation. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords hi-ctccchromatinconformationcaenorhabditis elegansgenomehttp://dx.doi.org/ /100004412human frontier science programlt000517/2011gabdank idan http://dx.doi.org/ /100000002national institutes of healthr01gm37706r01gm53804villeneuve anne m. fire andrew z. issue-copyright-statement© the author 2016 