Abstract
Resolving the molecular basis of a Mendelian condition remains challenging owing to the diverse mechanisms by which genetic variants cause disease. To address this, we developed a synchronized long-read genome, methylome, epigenome and transcriptome sequencing approach, which enables accurate single-nucleotide, insertion–deletion and structural variant calling and diploid de novo genome assembly. This permits the simultaneous elucidation of haplotype-resolved CpG methylation, chromatin accessibility and full-length transcript information in a single long-read sequencing run. Application of this approach to an Undiagnosed Diseases Network participant with a chromosome X;13-balanced translocation of uncertain significance revealed that this translocation disrupted the functioning of four separate genes (NBEA, PDK3, MAB21L1 and RB1) previously associated with single-gene Mendelian conditions. Notably, the function of each gene was disrupted via a distinct mechanism that required integration of the four ‘omes’ to resolve. These included fusion transcript formation, enhancer adoption, transcriptional readthrough silencing and inappropriate X-chromosome inactivation of autosomal genes. Overall, this highlights the utility of synchronized long-read multi-omic profiling for mechanistically resolving complex phenotypes.
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Data availability
All raw and processed sequencing data generated for GM12878, GM23485, GM20129 and HG02630 in this study have been submitted to the NCBI BioProject database (https://www.ncbi.nlm.nih.gov/bioproject/) under accession PRJNA1124997. Restrictions apply to the availability of some data generated or analyzed during this study to preserve subject confidentiality. Genomic data for our case report are accessible to the scientific community through the UDN (dbGaP). Individuals interested in accessing UDN data through dbGaP should submit a data access request. Detailed instructions for this process can be found on the NIH Scientific Data Sharing website (https://sharing.nih.gov/accessing-data/accessing-genomic-data/how-to-request-and-access-datasets-from-dbgap). Cell lines obtained from the National Institute of General Medical Sciences Human Genetic Cell Repository at the Coriell Institute for Medical Research include GM12878, GM23485, GM20129 and HG02630. The UDN318336 fibroblast line was obtained directly from UDN participant UDN318336. Hia5 enzyme and plasmid are available upon request. ONT data for the CpG comparison are available from Epi2me (https://labs.epi2me.io/giab-2023.05/, super accuracy analysis level 60× BAM files).
Processed FIRE results for GM12878, GM23485, GM20129, HG02630, UDN318336 and retinal organoid derived from UDN318336 are publicly available through https://doi.org/10.5281/zenodo.14511246, as well as through GitHub (https://github.com/StergachisLab/Fiber-seq-publication-data). Source data are provided with this paper.
Code availability
All software used in the study is publicly available. Specifically, the custom FIRE pipeline multi-ome-v0.1 (ref. 53; https://doi.org/10.5281/zenodo.12701477 and https://github.com/fiberseq/FIRE) and the custom k-mer-variant-phasing pipeline v0.0.1 (ref. 54; https://doi.org/10.5281/zenodo.10655504 and https://github.com/mrvollger/k-mer-variant-phasing) are publicly available and accessioned on Zenodo.
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Acknowledgements
We thank C. C. Lambert, A. Souppe, H. Dhillon and S. Zhang (all from PacBio) for assistance with library and sequencing preparations. A.B.S. holds a Career Award for Medical Scientists from the Burroughs Wellcome Fund and is a Pew Biomedical Scholar. This study was supported by the NIH (grants 1DP5OD029630 and 1U01HG010233), in addition to funds from the Collagen Diagnostic Laboratory, University of Washington, and the Brotman Baty Institute for Precision Medicine. Sequencing and data analysis were provided by the University of Washington Center for Rare Disease Research, with support from National Human Genome Research Institute (grants U01 HG011744 and U24 HG011746). Research from the UDN reported in this publication was supported by the National Institute of Neurological Disorders and Stroke of the NIH (awards U01HG010233, U01HG007942 and U01HG007530). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. M.R.V. was supported by a training grant (T32) from the NIH (2T32GM007454-46). The Eichler Lab provided the CH17-6D16 (chrX) and CH17-139G1 (chr13) BAC clones.
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Consortia
Contributions
A.B.S., M.R.V. and J.K. conceived the overall method design, coordinated activities from co-authors and wrote the first draft of the manuscript. M.R.V., E.S. and A.B.S. contributed to the analysis of Fiber-seq and MAS-seq data. K.C.E. and T.A.R. contributed to the generation of patient-derived retinal organoids, as well as the evaluation of RNA-seq data in retinal organoids. Y.S. contributed to the western blots, as well as mitochondrial functional studies. Y.M. created the Hia5 enzyme. E.E.B., S. Sheppeard, S. Strohbehn, S.M.S., E.A.R. and A.M.W. contributed to the analysis of genomic data. U.S. and P.H.B. contributed to the Sanger validation of the translocation breakpoints. K.M.D., G.P.J., F.M.H., M.J.B., A.L.Y., J.O., K.A.L., I.G., B.L.D., S.C. and A.A. contributed to patient phenotyping. M.H.-P. contributed to getting IRB approval for this study. J.R., A.B.S., K.C.E., M.K. and Y.H.C. contributed to the generation of Fiber-seq and mRNA samples. J.G.U. contributed to the mRNA concatenation protocol development and transcriptome data analysis. C.T.S. and A.M.W. contributed to CpG methylation calling and IGV visualization. I.J.M. and K.M.M. coordinated and performed the sequencing runs. All authors revised the manuscript.
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J.K., J.G.U., C.T.S., A.M.W., M.K. and I.J.M. are full-time employees at PacBio, a company developing single-molecule sequencing technologies. A.B.S. is a co-inventor on a patent relating to the Fiber-seq method (US17/995,058). The remaining authors declare no competing interests.
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Extended data
Extended Data Fig. 1 IGV view of integrated long-read multi-ome data.
IGV view showing an example genomic region for GM12878 (from top to bottom) the haplotype-resolved genome, CpG methylome, chromatin and full-length transcript annotations.
Extended Data Fig. 2 Haplotype-specific chromatin architectures.
Volcano plot showing the absolute difference in the percentage of chromatin fibers with chromatin accessibility for each peak genome-wide between the paternal and maternal haplotypes for GM12878, GM20129, HG02630 and GM24385. Peaks are divided into whether they are present on an autosome or the X chromosome, with the exception of GM24385, which is 46,XY. P value was calculated using a two-sided Fisher’s exact test without adjustment for multiple comparisons. Blue dash represents nominal significance line (p < 0.05), and red dash represents the Benjamini–Hochberg FDR correction significance line (FDR < 0.05). Peaks corresponding to known imprinted loci with nominally significant scores are denoted by red crosses.
Extended Data Fig. 3 Identification of haplotype-specific chromatin architectures within HLA locus.
a, View of single-molecule haplotype-resolved genetic information, CpG methylation information and chromatin accessibility information for a heterozygous single-nucleotide variant (SNP) identified for GM12878 within the HLA locus. Note that all information is derived from the same sequencing reads. Denoted below is the ___location of a predicted CTCF binding element, and immediately above it are two fibers that demonstrate single-molecule protein occupancy at this site. b, Quantification of CpG methylation surrounding this SNP (top), as well as the percentage of fibers with a FIRE element overlapping that SNP (bottom), by haplotype. The box bounds the interquartile range (IQR) divided by the median, and the whiskers extend to the minima and maxima of the data. *Top, p value = 0.00077 (one-sided paired t test without adjustment for multiple comparisons, n = 8). *Bottom, p value = 0.00061 (one-sided Fisher’s exact test without adjustment for multiple comparisons).
Extended Data Fig. 4 Haplotype-specific chromatin accessibility of NBEA promoter and transcript NMD.
a, Per-molecule chromatin accessibility of the NBEA promoter along both the der(13) and intact chr13 chromosomes within patient-derived fibroblasts. Chromatin accessibility was measured using Fiber-seq. b, Schematic representation for resolving whether the fusion transcripts are being subjected to nonsense-mediated decay (NMD). Specifically, long-read transcript sequencing was performed on samples before and after treatment with the NMD inhibitor cycloheximide. The libraries were sequenced to similar depths, and the total number of transcripts corresponding to the NBEA–chrX fusion transcripts was quantified in both samples. c, Same as b, but for the PDK3–MAB21L1 fusion transcript.
Extended Data Fig. 5 PDK3 protein levels in patient cells.
a, Western blot of phospho-PDH and PDH in HEK293 cells, as well as HEK293 cells containing a viral expression vector for the intact PDK3 protein, or the PDK3–MAB21L1 fusion protein. b, Western blot of PDK3 in HEK293 cells, as well as HEK293 cells containing a viral expression vector for the intact PDK3 protein, or the PDK3–MAB21L1 fusion protein. * indicates protein bands selectively present in the PDK3-MAB21L1 sample. c, Western blot of PDK3 and actin protein levels in patient-derived fibroblasts, as well as control age-matched fibroblast cultures and retinal organoids. All experiments in the figure were performed once.
Extended Data Fig. 6 Cell-selective expression of MAB21L1.
a, (Top) Bar plot showing the bulk tissue RNA expression of MAB21L1 (ENSG00000180660) from various GTEx samples are displayed by www.proteinatlas.org. (Bottom) Bulk expression of NBEA, MAB21L1 and PDK3 during retinal organoid differentiation as a function of the age of the organoid. TPM, transcripts per million. b, Volcano plot showing the absolute difference in the percentage of chromatin fibers with chromatin accessibility for each peak genome-wide between the paternal and maternal haplotypes for the retinal organoid Fiber-seq data from UDN318336. The MAB21L1 regulatory elements that are disrupted on der(13) are highlighted in red. Blue dash represents the nominal significance line (p < 0.05; two-sided Fisher’s exact test without adjustment for multiple comparisons).
Extended Data Fig. 7 3C chromatin interactions between PDK3 promoter and MAB21L1 enhancer.
Results from chromatin conformation capture (3C) experiment using induced pluripotent stem cells (iPSCs) and retinal organoids from UDN318336. For the iPSC 3C experiment, displayed is the relative enrichment of different regions of the PDK3 gene for interactions with the MAB21L1 enhancer. For the retinal organoid 3C experiment, as a limited amount of 3C library was obtained, displayed are the qualitative results of qPCR for each of the tested interactions.
Extended Data Fig. 8 PDK3 protein levels in patient retinal organoids and PHD activity in iPSCs.
a, Western blot images showing quantification of PDK3, phospho-PDH, PDH and β-actin levels in patient-derived retinal organoids, as well as age-matched retinal organoids from a separate unaffected and unrelated individual. Data from two separate protein extractions are displayed. Quantification of phospho-PDH to PDH level is shown at the bottom. b, Pyruvate dehydrogenase activity assay on iPSCs from UND318336, as well as control iPSCs. Data are presented as mean values ± SEM, and results are from three replicates (n = 3). NS, not significant (p value = 0.11), unpaired two-sided t test.
Extended Data Fig. 9 Comparison with prior chromosome X;13 translocations.
Ideogram showing the translocation breakpoints and derivative chromosomes for this case, as well as a previously published case who similarly had bilateral retinoblastomas. The translocation breakpoints for the previously published case are estimated based on the karyotype.
Extended Data Fig. 10 Allelic imbalance differences along chr13.
a, Swarm plot showing the overall haplotype imbalance in chromatin accessibility along autosomes (except for chromosomes 13 and 14), chromosome X, chromosome 14 and two portions of chromosome 13 within GM12878 and GM24385 cells. P value calculated using a one-sided Mann–Whitney U test without adjustments for multiple comparisons. b, Swarm plot showing the overall haplotype imbalance in transcript production along autosomes (except for chromosomes 13 and 14), chromosome X, chromosome 14 and two portions of chromosome 13 within UDN318336 and GM12878 cells. P value calculated using a one-sided Mann–Whitney U test without adjustments for multiple comparisons. c, Swarm plot showing the haplotype imbalance in chromatin accessibility at different regions of chromosome 13 in fibroblasts from UDN318336. P value calculated using a one-sided Mann–Whitney U test without adjustments for multiple comparisons.
Supplementary information
Supplementary Information
Supplementary Tables 1–3.
Supplementary Table 4
Complete lists of consortia members.
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Vollger, M.R., Korlach, J., Eldred, K.C. et al. Synchronized long-read genome, methylome, epigenome and transcriptome profiling resolve a Mendelian condition. Nat Genet 57, 469–479 (2025). https://doi.org/10.1038/s41588-024-02067-0
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DOI: https://doi.org/10.1038/s41588-024-02067-0
