bioRxiv preprint doi: https://doi.org/10.1101/2020.05.20.105379; this version posted May 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Massively multiplex single-molecule oligonucleosome footprinting 2 3 Nour J Abdulhay1#, Colin P McNally1#, Laura J Hsieh1, Sivakanthan Kasinathan2, Aidan Keith1, Laurel S 4 Estes1, Mehran Karimzadeh1,3, Jason G Underwood4, Hani Goodarzi1,5, Geeta J Narlikar1, Vijay Ramani1,5* 5 6 1 Department of Biochemistry & Biophysics, University of California San Francisco, San Francisco CA 7 2 Department of Pediatrics, Stanford University, Palo Alto, CA 8 3 Vector Institute, Toronto, ON, Canada 9 4 Pacific Biosciences of California Inc, Menlo Park, CA 10 5 Bakar Computational Health Sciences Institute, San Francisco, CA 11 12 # These authors contributed equally 13 * To whom correspondence may be addressed: [email protected] 14 15 ABSTRACT 16 Our understanding of the beads-on-a-string arrangement of nucleosomes has been built largely on high- 17 resolution sequence-agnostic imaging methods and sequence-resolved bulk biochemical techniques. To bridge 18 the divide between these approaches, we present the single-molecule adenine methylated oligonucleosome 19 sequencing assay (SAMOSA). SAMOSA is a high-throughput single-molecule sequencing method that 20 combines adenine methyltransferase footprinting and single-molecule real-time DNA sequencing to natively 21 and nondestructively measure nucleosome positions on individual chromatin fibres. SAMOSA data allows 22 unbiased classification of single-molecular 'states' of nucleosome occupancy on individual chromatin fibres. 23 We leverage this to estimate nucleosome regularity and spacing on single chromatin fibres genome-wide, at 24 predicted transcription factor binding motifs, and across both active and silent human epigenomic domains. 25 Our analyses suggest that chromatin is comprised of a diverse array of both regular and irregular single- 26 molecular oligonucleosome patterns that differ subtly in their relative abundance across epigenomic domains. 27 This irregularity is particularly striking in constitutive heterochromatin, which has typically been viewed as a 28 conformationally static entity. Our proof-of-concept study provides a powerful new methodology for studying 29 nucleosome organization at a previously intractable resolution, and offers up new avenues for modeling and 30 visualizing higher-order chromatin structure. 31 32 1-sentence summary: High-throughput single-molecule real-time footprinting of chromatin arrays reveals 33 heterogeneous patterns of oligonucleosome occupancy. 34 35 Keywords: chromatin; nucleosome positioning; single-molecule sequencing; gene regulation 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.20.105379; this version posted May 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 INTRODUCTION 2 The nucleosome is the atomic unit of chromatin. Nucleosomes passively and actively template the 3 majority of nuclear interactions essential to life by determining target site access for transcription factors1, 4 bookmarking active and repressed chromosomal compartments via post-translational modifications2, and 5 safeguarding the genome from mutational agents3. Our earliest views of the beads-on-a-string arrangement of 6 chromatin derived from classical electron micrographs of chromatin fibres4, which have since been followed 7 by both light5 and electron microscopy6,7 studies of in vitro-assembled and in vivo chromatin. In parallel, 8 complementary biochemical methods using nucleolytic cleavage have successfully mapped the subunit 9 architecture of chromatin structure at high resolution. These cleavage-based approaches can be stratified into 10 those that focus primarily on chromatin accessibility8 (i.e. measuring ‘competent’ active chromatin9), and those 11 that survey nucleosomal structure uniformly across active and inactive genomic compartments. Understanding 12 links between chromatin and gene regulation requires sensitive methods in all three of these broad categories: 13 in this study we advance our capabilities in the third, focusing on a novel method to map oligonucleosomal 14 structures genome-wide. 15 Nucleolytic methods for studying nucleosome positioning have historically used cleavage reagents 16 (e.g. dimethyl sulphate10, hydroxyl radicals11, nucleases12) followed by gel electrophoresis and / or Southern 17 blotting to map the abundance, accessibility, and nucleosome repeat lengths (NRLs) of chromatin fibers13. 18 More recently, these methods have been coupled to high-throughput short-read sequencing14, enabling 19 genome-wide measurement of average nucleosome positions. While powerful, all of these methods share key 20 limitations: measurement of individual protein-DNA interactions inherently requires destruction of the 21 chromatin fibre and averaging of signal across many short molecules. These limitations extend even to single- 22 molecule methyltransferase-based approaches15-17, which have their own biases (e.g. CpG / GpC bias; presence 23 of endogenous m5dC in mammals; DNA damage due to bisulphite conversion), and are still subject to the 24 short- length biases of Illumina sequencers. While single-cell18,19 and long-read single-molecule20 genomic 25 approaches have captured some of this lost contextual information, single-cell data are generally sparse and 26 single-molecule Array-seq data must be averaged over multiple molecules. Ultimately, these limitations have 27 hindered our understanding of how combinations of ‘oligonucleosomal patterns’ (i.e. discrete states of 28 nucleosome positioning and regularity on single DNA molecules) give rise to active and silent chromosomal 29 domains. 30 The advent of third-generation (i.e. high-throughput, long-read) sequencing offers a potential solution 31 to many of these issues21. Here, we demonstrate Single-molecule Adenine Methylated Oligonucleosome 32 Sequencing Assay (SAMOSA), a method that combines adenine methyltransferase footprinting of 33 nucleosomes with base modification detection on the PacBio single-molecule real time sequencer22 to measure 34 nucleosome positions on single chromatin templates. We first present proof-of-concept of SAMOSA using 35 gold-standard in vitro assembled chromatin fibres, demonstrating that our approach captures single-molecule 36 nucleosome positioning at high-resolution. We next apply SAMOSA to oligonucleosomes derived from K562 37 cells to profile single-molecule nucleosome positioning genome-wide. Our data enables unbiased classification 38 of oligonucleosomal patterns across both euchromatic and heterochromatic domains. These patterns are 39 influenced by multiple epigenomic phenomena, including the presence of predicted transcription factor 40 binding motifs and post-translational histone modifications. Consistent with estimates from previous studies, 41 our approach reveals enrichment for long, regular chromatin arrays in actively elongating chromatin, and 42 highly accessible, disordered arrays at active promoters and enhancers. Surprisingly, we also observe a large 43 amount of heterogeneity within constitutive heterochromatin domains, with both mappable H3K9me3- 44 decorated regions and human major satellite sequences harboring a mixture of irregular and short-repeat-length 45 oliognucleosome types. Our study provides a proof-of-concept framework for studying chromatin at single- 46 molecule resolution while suggesting a highly dynamic nucleosome-DNA interface across chromatin sub- 47 compartments. 48 49 RESULTS 50 Single-molecule real-time sequencing of adenine-methylated chromatin captures nucleosome footprints 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.20.105379; this version posted May 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Existing methyltransferase accessibility assays either rely on bisulfite conversion15-17 or use the Oxford 2 Nanopore platform to detect DNA modifications23-25. We hypothesized that high-accuracy PacBio single- 3 molecule real-time sequencing could detect m6dA deposited on chromatin templates to natively measure 4 nucleosome positioning. To test this hypothesis, we used the nonspecific adenine methyltransferase EcoGII26 5 to footprint nonanucleosomal chromatin arrays generated through salt-gradient dialysis (Supplementary 6 Figure 1), using template DNA containing 9 tandem repetitive copies of the Widom 601 nucleosome 7 positioning sequence27 separated by ~46 basepairs (bp) of linker sequence followed by ~450 bp of sequence 8 without any known intrinsic affinity for nucleosomes. After purifying DNA, polishing resulting ends, and 9 ligating on barcoded SMRTBell adaptors, we subjected libraries to sequencing on PacBio Sequel or Sequel II 10 flow cells, using unmethylated DNA and methylated naked DNA as controls (Figure 1A). After filtering low 11 quality reads, we analyzed a total of 33,594 single molecules across all three conditions. Across both platforms,