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3D mapping and accelerated super-resolution imaging of the human genome using in situ sequencing

Nature Methods volume 17, pages 822–832 (2020)Cite this article

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Abstract

There is a need for methods that can image chromosomes with genome-wide coverage, as well as greater genomic and optical resolution. We introduce OligoFISSEQ, a suite of three methods that leverage fluorescence in situ sequencing (FISSEQ) of barcoded Oligopaint probes to enable the rapid visualization of many targeted genomic regions. Applying OligoFISSEQ to human diploid fibroblast cells, we show how four rounds of sequencing are sufficient to produce 3D maps of 36 genomic targets across six chromosomes in hundreds to thousands of cells, implying a potential to image thousands of targets in only five to eight rounds of sequencing. We also use OligoFISSEQ to trace chromosomes at finer resolution, following the path of the X chromosome through 46 regions, with separate studies showing compatibility of OligoFISSEQ with immunocytochemistry. Finally, we combined OligoFISSEQ with OligoSTORM, laying the foundation for accelerated single-molecule super-resolution imaging of large swaths of, if not entire, human genomes.

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Fig. 1: Using OligoFISSEQ to sequence barcoded Oligopaints in situ.
Fig. 2: OligoFISSEQ-LIT on 36plex-5K.
Fig. 3: Every-pixel analysis pipeline on 36plex-5K.
Fig. 4: Improving O-LIT by using JEB.
Fig. 5: Tracing 46 regions along the X chromosome.
Fig. 6: OligoFISSEQ extensions and applications.

Data availability

All data are available in the main text or the supplementary materials, and materials are available upon request. Information regarding all datasets (for example, cells, replicates and filters) can be found in Supplementary Table 9. Source data are provided with this paper.

Code availability

All code is available at https://github.com/3DGenomes/OligoFISSEQ/.

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Acknowledgements

We acknowledge members of the Marti-Renom and Wu laboratories for technical and conceptual support, especially T. Ryu, A. Lioutas and S. Aufmkolk as well as J. AlHaj Abed, S. D. Lee, J. Erceg and T. Hatkevich; B. Beliveau, H. Sasaki, J. Horrell, L. Cai, J. Kishi and P. Soler-Vila for discussion; D. Barclay, R. Kohman, E. Iyer, K. Rodgers, A. Skrynnyk, J. Tam and R. Terry for discussion about FISSEQ and sequencing reagents; S. Alon, F. Chen, Z. Chiang, D. Goodwin, A. Payne, A. Sinha and O. Wassie for discussion about FISSEQ; C. Ebeling, J. Rosenberg and J. Stuckey for discussion and technical assistance; F. Pan and A. Hutchinson for assistance in procuring SOLiD reagents; P. Montero-Llopis and the MicRoN imaging core at Harvard Medical School; the ImageJ discussion forum; and StackOverflow. This work was supported by a Damon Runyon Dale F. Frey Breakthrough Award (to B.J.B.) to support B.J.B. and E.A.H., awards from the NSERC of Canada (PGS D) to P.L.R., the NIH (HG005550 and HG008525) and NSF (DGE1144152) to E.R.D., the European Research Council under the Seventh Framework Program (FP7/2007–2013 609989), the European Union’s Horizon 2020 Research and Innovation Program (676556) and the Spanish Ministerio de Ciencia, Innovación y Universidades (BFU2017-85926-P) to M.A.M.-R., the Centro de Excelencia Severo Ochoa 2013–2017 (SEV-2012-0208) and the CERCA Programme/Generalitat de Catalunya to the CRG, from the NIH to GMC (RM1HG008525-03) and the NIH (DP1GM106412, R01HD091797 and R01GM123289) to C.-t.W.

Author information

Author notes

  1. Son C. Nguyen

    Present address: Department of Genetics, University of Pennsylvania, Philadelphia, PA, USA

  2. Evan R. Daugharthy

    Present address: ReadCoor, Cambridge, MA, USA

  3. These authors contributed equally: Huy Q. Nguyen, Shyamtanu Chattoraj, David Castillo.

Authors and Affiliations

  1. Department of Genetics, Harvard Medical School, Boston, MA, USA

    Huy Q. Nguyen, Shyamtanu Chattoraj, Son C. Nguyen, Guy Nir, Antonios Lioutas, Nuno M. C. Martins, Paul L. Reginato, Mohammed Hannan, George M. Church, Evan R. Daugharthy & C.-ting Wu

  2. CNAG-CRG, Centre for Genomic Regulation (CRG), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain

    David Castillo & Marc A. Marti-Renom

  3. Wyss Institute, Harvard Medical School, Boston, MA, USA

    Guy Nir, Paul L. Reginato, George M. Church, Evan R. Daugharthy & C.-ting Wu

  4. Department of Genome Sciences, University of Washington, Seattle, WA, USA

    Elliot A. Hershberg & Brian J. Beliveau

  5. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Paul L. Reginato

  6. Brotman Baty Institute for Precision Medicine, Seattle, WA, USA

    Brian J. Beliveau

  7. Department of Systems Biology, Harvard Medical School, Boston, MA, USA

    Evan R. Daugharthy

  8. ReadCoor, Cambridge, MA, USA

    Evan R. Daugharthy

  9. CRG, BIST, Barcelona, Spain

    Marc A. Marti-Renom

  10. Pompeu Fabra University, Barcelona, Spain

    Marc A. Marti-Renom

  11. ICREA, Barcelona, Spain

    Marc A. Marti-Renom

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  1. Huy Q. Nguyen
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Contributions

H.Q.N., S.C., D.C., S.C.N., G.M.C., E.R.D., M.A.M.R. and C.-t.W. conceived the study with the original conceptualization of OligoFISSEQ contributed by S.C.N and E.R.D.; G.N., A.L. and N.M.C.M. provided guidance for barcode design and angle analysis; A.L., E.A.H. and B.J.B. provided guidance for Oligopaint sequences and barcode design. P.L.R. supported early protocol development; M.H. provided technical support; H.Q.N. and S.C. designed and performed the experiments. H.Q.N., S.C., D.C., G.M.C., M.A.M.R. and C.-t.W. analyzed the data; H.Q.N. wrote the manuscript with S.C., D.C., M.A.M.R. and C.-t.W. with input from all authors; C.-t.W. oversaw the project.

Corresponding authors

Correspondence to Marc A. Marti-Renom or C.-ting Wu.

Ethics declarations

Competing interests

Harvard University has filed patent applications on behalf of C.-t.W., H.Q.N. and S.C., pertaining to Oligopaints and related oligonucleotide-based methods for genome imaging. E.R.D. is currently an employee of ReadCoor and has an equity interest in ReadCoor. Potential conflicts of interest for G.M.C. are listed on http://arep.med.harvard.edu/gmc/tech.html/. C.-t.W. has an equity interest in ReadCoor and an active research collaboration with Bruker Nano in her laboratory at Harvard Medical School.

Additional information

Peer review information Lei Tang was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Chr19-20K and 36plex-5K-O-LIT optimization.

a, Chr19-20K targets 18,536 Oligopaint oligos to human chromosome 19. Right, Chr19-20K detection with secondary oligo (red) in PGP1f cells representative of 5 replicates. b, Signal is completely removed in each OligoFISSEQ method after cleavage. Images showing two rounds of sequencing with a cleavage step (C) and representative of 4 replicates. c, 36plex-5K O-LIT off of both Mainstreet and Backstreet (MSBS; bottom, red) produces stronger signal than off of Mainstreet (MS; top, blue). Cy5 channel from first round of O-LIT. n = 1. d, O-LIT off of both streets produces stronger signal than off of MS. Grey intensity value measurements from yellow lines in panel c. n = 1. e, Raw, non-deconvolved field of view of cell from Figs. 2c, d and 3a–c. Maximum z-projection. n = 1. f, Manual decoding of cell from panel c and Figs. 2c, d and 3a–c yields 100% target recovery. n = 1. g, Tier1 detection efficiency after 36plex-5K O-LIT off of both streets and detected with TrackMate (blue, 29.93 ± 4.9%) or Every-pixel (orange, 62.8% ± 4.8%). n = 111 cells from 3 replicates. Detection efficiency from individual replicates are plotted. Error bars represent 95% bootstrap confidence interval of the mean.

Source data

Extended Data Fig. 2 Detection efficiency after 36plex-5K O-LIT.

a, Detection efficiency without filtering after 36plex-5K O-LIT off of both streets. 95 ± 5.15% of targeted regions are detected (n = 611 from 15 replicates). Detection efficiency from individual replicates are plotted. Error bars represent 95% bootstrap confidence interval of the mean. b, False positive (FP) discovery rate from panel a. FP discovery rate from individual replicates are plotted. Error bars represent 95% bootstrap confidence interval of the mean. c, Tier 1 detection efficiency after 36plex-5K O-LIT off of Mainstreet (orange, 61.93 ± 12%, n = 53 from 2 replicates) versus off of both streets (blue, 62.17% ± 6.68%, n = 611 cells from 15 replicates). Detection efficiency from individual replicates are plotted. Error bars represent 95% bootstrap confidence interval of the mean. d, FP discovery rate from panel c. Using Mainstreet = 8.64% and using both streets = 5.29%. FP discovery rate from individual replicates are plotted. Error bars represent 95% bootstrap confidence interval of the mean. e, Tier 2 detection efficiency after 36plex-5K off of Mainstreet (orange, 92.3% ± 3.42% from 53 cells from 2 replicates) versus off of both streets (blue, 80.19 ± 7.29%, n = 611 cells from 15 replicates). Detection efficiency from individual replicates are plotted. Error bars represent 95% bootstrap confidence interval of the mean. f, Detection efficiency after 36plex-5K O-LIT off of both streets for individual cells from 15 replicates in panel e. g, Percentage of cells displaying a range of efficiencies of barcode detection after 36plex-5K O-LIT off of both streets. Data taken from panel e. h, Principal component analysis showing lack of batch effect in 36plex datasets (n = 1171 cells from 15 36plex-5K O-LIT replicates and 8 36plex-1K O-eLIT replicates).

Source data

Extended Data Fig. 3 O-LIT with 36plex-5K to interrogate genome organization.

a, Chromosome traces of Cell 611 after Tier 2 detection of cell 611 after four rounds of O-LIT 36plex-5K off of both streets. 59/66 (89%) of 36plex-5K targets were detected. Image is from the first round of O-LIT with target identities. n = 1. b, Ball and stick of Cell 611. Colored spheres represent chromosomal targets, while black spheres represent targets that were not detected and, thus, were placed by calculating the median proportionate distance between flanking detected targets. Beginning of chromosome (for example 2pR1) marked by an asterisk. c, Single-cell pairwise spatial distance matrix after Tier 1 (top) and Tier 2 (bottom) detection of the nucleus in Fig. 3. Targets are represented on the x-axis with homologs separately displayed. Undetected targets are represented by grey lines. d, Single-cell pairwise spatial distance matrix after Tier 1 (top) and Tier 2 (bottom) detection of Cell 611. Targets are represented on the x-axis with homologs separately displayed. Undetected targets are represented by grey lines. e, 36plex-5K population pairwise spatial distances (top, from Fig. 3f). Average pairwise spatial distances from cell population after Tier 1 detection (n = 611 from 15 replicates). (Spearman’s rank correlation 0.705, two-sided p-value for a hypothesis test whose null hypothesis is that two sets of data are uncorrelated = 1.77e-174). Measurements from homologous targets were combined. Bottom, Hi-C data of 36plex-5K targets obtained from (Nir et al. 2018). f, Average distances between the nuclear membrane and the closest of the six targets imaged for each chromosome. (n = 686, 668, 364, 586, 760, and 494 for Chr2, 3, 5, 16, 19, and X, respectively.) The thick line in each violin plot represents the Interquartile range (IQR), the white dot marks the median and the thin lines extend 1.5 times the IQR.

Source data

Extended Data Fig. 4 O-LIT with 36plex-5K to interrogate homolog organization.

a, Minimum distances between heterologous and homologous chromosomes. All measurements represent distances between the geometric centers of chromosomes for which all six targets were imaged. Distances between a chromosome and a heterologous chromosome is the shorter of the two distances between that chromosome and the two homologous copies of the heterologous chromosome (n = 686, 668, 364, 586, 760, and 494 for Chr2, 3, 5, 16, 19, and X, respectively). Inter-homolog distances for Chr16 and 19 are less than those for Chr2, 3, and 5 (independent-samples t-test p = 4.28 ×10-37). Boxes represent the IQR (25th, 50th and 75th percentiles) and whiskers extend 1.5 times the IQR. b, Number of cells with varying numbers of homologs split by K-means clustering. The K-means algorithm was applied to 258 nuclei, individually, to cluster chromosomes into two groups based on proximity and then report the number of homolog pairs that were split by the clustering. A value of “5” indicates that the homologs from each five pairs of imaged autosomes in a single nucleus clustered into two spatially separate groups. Observed, PGP1f cells. Directed random, raw positions in Observed but with the chromosome identities of all positions randomized, with the larger chromosomes (2, 3, 5) biased towards the nuclear periphery and smaller chromosomes (16 and 19) biased towards the nuclear interior. Completely random category, randomization of the chromosome identities carried out with no spatial bias. The significance of each pair was evaluated from a two proportion z-test with n = 258 for each category with a null hypothesis of equal proportion and a significance level of 0.05. c, Density plots of homolog positions. Built by using Kernel density estimation (KDE) of nuclei projected and aligned along the x-y plane of the position of the chromosomes. d, Pie charts of total number of cells with homologs split by a virtual line along the y-axis. e, Number of aligned cells with homologs split by a virtual line parallel to the y-axis at different distances from the origin, that is, number of autosomes with one of their homologs on the left of the line and the other on the right (n = 258 for each category). Boxes represent the IQR (25th, 50th and 75th percentiles) and whiskers extend 1.5 times the IQR.

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Extended Data Fig. 5 O-eLIT with JEB.

a, Chr19-9K. One round of O-LIT (SOLiD) or O-eLIT (JEB) off of Mainstreet. Maximum z-projections representative of 2 replicates. b, Chr19-9K signal over nuclear background measurements after one round of O-LIT (orange; n = 113 puncta from 55 cells from 2 replicates) or O-eLIT (blue; n = 136 puncta from 57 cells from 2 replicates). Bar is the mean and SD. c, Tier 1 detection of 36plex-1K after five rounds of O-LIT with SOLiD reagents (orange; average of 51.75%, n = 41) or O-eLIT with JEB (blue; average of 61.2 ± 10.2%, n = 440 from 9 replicates). Detection efficiency from individual replicates are plotted. Error bars represent 95% bootstrap confidence interval of the mean. 36plex-1K library shares first 1,000 Oligopaint oligos of each target in 36plex-5K. For example, for target 2pR1, 36plex-5K spans the chromosomal region from nt position 1,002,895 to 1,660,898 (~658 kb), whereas 36plex-1K spans the region from nt 1,002,895 to 1,147,495 (~144 kb). d, FP discovery rate from panel c. SOLiD = 7.49% and JEB = 8.95%. FP discovery rate from individual replicates are plotted. Error bars represent 95% bootstrap confidence interval of the mean. e, Chromosome traces and ball and stick of Fig. 4c cell after Tier 2 detection and five rounds of O-eLIT 36plex-1K. 63/66 (95%) targets were detected. Asterisks, beginning of chromosomes. n = 1. f, Single-cell pairwise spatial distance matrices of panel C cell. g, 36plex-1K population pairwise spatial distance measurements (top, from Fig. 3f). Average pairwise spatial distance from cell population after Tier 1 detection (n = 440 from 9 replicates). Measurements from homologous targets were combined. Bottom, Hi-C data of 36plex-5K targets obtained from (Nir et al. 2018). h, 36plex-1K detection rate for individual cells from 9 replicates. i, Percentage of cells displaying a range of efficiencies of barcode detection after 36plex-1K O-eLIT off of Mainstreet.

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Extended Data Fig. 6 O-eLIT with ChrX-46plex-2K.

a, ChrX-46plex-2K O-eLIT Tier 1 detection off of one street and off of both streets combined (52.86 ± 5.78% from 177 cells from 7 replicates). Detection efficiency from individual replicates are plotted. Error bars represent 95% bootstrap confidence interval of the mean. b, FP discovery rate from panel a. Error bars represent 95% bootstrap confidence interval of the mean. c, Single-cell pairwise spatial distance matrix after Tier 1 (top) and Tier 2 (bottom) detection of Cell 1 from Fig. 5b. Undetected targets are represented by grey lines. d, Chromosome traces (top) and ball and stick representation (bottom) of Cell 177 after Tier 2 detection and interpolation and five rounds of O-eLIT on ChrX-46plex-2K off of both streets. Image is from the first round of O-eLIT with target identities. n = 1. e, Single-cell pairwise spatial distance matrix after Tier 1 (top), Tier 2 (bottom) of Cell 177 (left), and Tier 2 (top) and interpolation (bottom) of same cell (right). Undetected targets are represented by grey lines. f, ChrX-46plex-2K population pairwise spatial distances (top). Average pairwise spatial distances from cell population after Tier 1 detection (n = 177 from 7 replicates). Bottom, Hi-C (Nir et al. 2018) data of ChrX-46plex-2K targets. (Spearman’s rank correlation 0.641, two-sided p-value for a hypothesis test whose null hypothesis is that two sets of data are uncorrelated = 7.074e-245). g, ChrX-46plex-2K detection rate for individual cells from 7 replicates. h, Percentage of cells displaying a range of efficiencies of barcode detection after ChrX-46plex-2K O-eLIT. i, Mean spatial distance versus Interaction frequency of Hi-C (Nir et al. 2018) of ChrX-46plex-2K targets. Pearson correlation coefficient (r = −0.84) and p-value = 5.08E-275 (two-sided, using slope = 0 for null hypothesis and Wald Test with t-distribution as test statistic) of the linear least-squares regression. j, Mean spatial distance versus genomic distance for all pairwise ChrX-46plex-2K targets (n = 177 from 7 replicates).

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Extended Data Fig. 7 O-eLIT identifies clusters after ChrX-46plex O-eLIT.

a, Hierarchical clustering based on structure of ChrX traces from ChrX-46plex after 5 rounds of O-eLIT and Tier 2 detection yielded two clusters (Cluster 1 = 20; Cluster 2 = 156). See Methods for more details. b, ChrX representative models (existing traces that are closer to the virtual centroid) of the two clusters obtained after Hierarchical clustering in panel a. c, ChrX-46plex-2K population contact matrix of two clusters derived after Hierarchical clustering in panel a where pairwise spatial distances are considered to be in contact if less than 2 µm apart. d, Radius of gyration for the two clusters (Cluster 1 = 20; Cluster 2 = 156) derived after the hierarchical clustering shown in panel a. The thick line in each violin plot represents the Interquartile range (IQR), the white dot marks the median and the thin lines extend 1.5 times the IQR.

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Extended Data Fig. 8 Angles from 36plex.

a, Measurements of angles formed by three points along the p arm (left), q arm (right), and intersection of vectors formed by pR2-pR3 and qR1-qR2 (middle) for each chromosome. Measurements were obtained by combining data from 36plex-5K and 36plex-1K analyses and selecting chromosomes that had all six targets identified. Chr2: n = 686, Chr3: n = 668, Chr5: n = 363, Chr16: n = 586, Chr19: n = 760, ChrX: n = 493 (n = 1,051 cells from 24 replicates; for 36plex-5K, n = 611 from 15 replicates; for 36plex-1K, n = 440 from 9 replicates). b, Distribution of angles formed by segments in panel a. The thick line in each violin plot represents the Interquartile range (IQR), the white dot marks the median and the thin lines extend 1.5 times the IQR. c, Box plots comparing p and q arm angles. Two-sided student’s t-test with null hypothesis of equal mean was performed to compare arms, ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Boxes represent the IQR (25th, 50th and 75th percentiles) and whiskers extend 1.5 times the IQR. Sample size information in a). Exact p-values for each chromosome: Chr.2 = 4.149e-16, Chr.3 = 0.004, Chr.5 = 0.093, Chr.16 = 1.357e-14, Chr.19 = 3.325e-11, Chr.X = 0.101. d, Linear least-squares regression between arm angle and arm length with Pearson correlation coefficient r = 0.26 and p-value = 0.42 (two-sided, using slope = 0 for null hypothesis and Wald Test with t-distribution as test statistic).

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Extended Data Fig. 9 O-eLIT comparison of X chromosomes in female IMR-90 cells after ChrX-46plex-2K O-eLIT off of both streets.

a, First round of O-eLIT sequencing. MacroH2A.1 immunostaining after five rounds of O-eLIT marks the Xi. n = 1. b, c, Xi and Xa traces (b) and ball and stick (c) of panel a nucleus after Tier 2 analysis and interpolation of missing targets. Sphere color corresponds to chromosome cartoon. n = 1. d, Single-cell pairwise spatial distances after interpolation of missing targets in panel a. e, Tier 2 target detection efficiency after five rounds of O-eLIT. 38.57% of targeted regions are detected in 71 cells. Detection efficiency from individual replicates are plotted. Error bars represent 95% bootstrap confidence interval of the mean. f, Population pairwise spatial distances after Tier 1 detection (n = 71 cells) and Hi-C data of IMR-90 cells (Rao et al. 2014). g, Population contact maps (top) where two targets are considered to be in contact if less than 2 µm apart (n = 315 chromosomes). Bottom, Hi-C data as in panel f. (Spearman’s rank correlation with the Hi-C matrix is r = 0.733, two-sided p-value for a hypothesis test whose null hypothesis is that two sets of data are uncorrelated = 2.564 ×10e-175). h, Radius of gyration for the Xi (n = 40 chromosomes) and Xa (n = 31 chromosomes). The thick line in each violin plot represents the Interquartile range (IQR), the white dot marks the median and the thin lines extend 1.5 times the IQR. P-value = 7.08 ×10e-6 (two-sided t-test whose null hypothesis is equal means). i, j, Linear plot of the mean spatial distance versus the genomic distance for all pairwise targets for Xi (n = 40 chromosomes) and Xa (n = 31 chromosomes). k–l, Population contact maps for Xi (n = 40 chromosomes) and Xa (n = 31 chromosomes) with eigenvector analysis used to identify different domains. X1-X18 (white) and X19-X46 (grey) targets p and q arms, respectively.

Source data

Extended Data Fig. 10 OligoFISSEQ applications.

a, O-eLIT and immunofluorescence (IF). 36plex-1K was sequenced 5 rounds with O-eLIT off Mainstreet. Then, the same sample was prepared for IF and stained with antibodies. Samples were counterstained with wheat germ agglutinin (WGA) to stain membranes. Images are from deconvolved, maximum z-projections representative of 2 replicates. b, Chromosomal regions imaged with OligoSTORM from Fig. 6d enlarged and displayed separately. Orientation may differ from Fig. 6d. n = 1. c, 8 rounds of O-LIT sequencing of Chr19-9K off of Mainstreet. Images are maximum z-projections. Signal is detectable in all rounds even though the imaging was conducted without the advantage of eLIT, suggesting that 8 rounds of O-eLIT will produce even stronger signals. Images are representative of 2 replicates. d, O-LIT is compatible with gel embedding and target amplification via rolling circle amplification (RCA). Chr19-9K was hybridized to PGP1f cells, after which the sample was embedded in a hydrogel and then cleared of cellular background with proteinase. Next, a molecular inversion probe (MIP) was hybridized to a Chr19-9K specific barcode on Backstreet as well as a fluorophore labeled (purple) secondary oligo to Mainstreet to visualize Chr19-9K Oligopaint oligos. MIPs were circularized via ligation and RCA, after which the first digit of the barcode was sequenced using O-LIT (green). Images are representative of two replicates. e, Comparison of secondary fluorophore signal (2o) versus first round sequencing signal (LIT) from puncta in panel b images. Center values are mean values (3.4 for 2o and 4.9 for O-LIT) with SD.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–4, Supplementary Notes 1 and 2 and Supplementary Protocols 1–5.

Reporting Summary

Supplementary Tables 1, 7 and 8

Lists of reagents, oligonucleotides and O-HIT secondary oligonucleotides used in this study.

Supplementary Tables 2–6 and 16

Oligopaint library sequences for all libraries used in this study.

Supplementary Table 9–11

Image dataset information.

Supplementary Table 12

Genomic and probe information for all libraries used in this study.

Supplementary Table 13

List of Spearman’s rank correlations and two-sided P values for a hypothesis test whose null hypothesis is that two sets of data are uncorrelated.

Supplementary Table 14

Every-pixel analysis intensity thresholds.

Supplementary Table 15

Mean and s.d. of the distance from the center of chromosomes to the nuclear envelope.

Source data

Source Data Fig. 1

O-LIT, O-SIT and O-HIT barcode detection for Chr19-20K library.

Source Data Fig. 3

36plex-5K data.

Source Data Fig. 4

36plex-1K data.

Source Data Fig. 5

ChrX-46plex-2K data.

Source Data Fig. 6

Six-gene library detection data.

Source Data Extended Data Fig. 1

36plex-5K: street sequencing, manual tracing and every-pixel comparison with TrackMate.

Source Data Extended Data Fig. 2

36plex-5K detection data.

Source Data Extended Data Fig. 3

36plex-5K extra cell traces and genome organization comparison.

Source Data Extended Data Fig. 4

36plex chromosome clustering data.

Source Data Extended Data Fig. 5

36plex-1K extra data.

Source Data Extended Data Fig. 6

ChrX-46plex-2K extra data.

Source Data Extended Data Fig. 7

ChrX cluster data.

Source Data Extended Data Fig. 8

36plex angle data.

Source Data Extended Data Fig. 9

ChrX-46plex-2K in IMR-90 data.

Source Data Extended Data Fig. 10

Intensity measurements after rolling circle amplification of Chr19-9K.

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Nguyen, H.Q., Chattoraj, S., Castillo, D. et al. 3D mapping and accelerated super-resolution imaging of the human genome using in situ sequencing. Nat Methods 17, 822–832 (2020). https://doi.org/10.1038/s41592-020-0890-0

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