calibration beads) were sorted onto a transparent plastic plate cover for alignment calibration. conformation maps from 14 cortical cell types. These datasets reveal the genome-wide association between cell-type specific chromatin conformation and differential DNA methylation, Salidroside (Rhodioloside) suggesting pervasive interactions between epigenetic processes regulating gene expression. Introduction Three-dimensional genome architecture is a critical feature of gene regulation in metazoan organisms 1-3. Chromatin conformation profiling has revealed the presence of features such as Topologically Associated Domains (TADs) and enhancer-promoter interactions 4-9. Despite the increasing utility of these datasets, most existing chromatin conformation maps are generated from cell lines or from bulk tissues 4,8-11. While these data has helped to elucidate general principles of chromatin business, it cannot fully represent the diversity of cell types that arise 3C experiments 8, 18, 19. The ligated 3C nuclei are dispensed into 384 well PCR plates using Fluorescence-activated nuclei sorting (FANS) and subject to proteinase digestion and bisulfite conversion , and libraries are constructed similar to our previous snmC-seq2 method (Fig. 1) 16,20 . When performed as a bulk assay (m3C-seq) ligated nuclei are not sorted into well but treated in bulk. Open in a separate window Physique 1. Outline of the single-nucleus methyl-3C sequencing (sn-m3C-seq) method.Samples are processed with a typical in situ 3C/Hi-C procedure following by single-cell DNA methylome library preparation using snmC-seq2. To evaluate the quality of chromatin contact maps generated by m3C-seq, we compared bulk m3C-seq data to conventional bulk 3C-seq and Hi-C profiles in mESCs. Both Hi-C/3C and bisulfite conversion present challenges for Salidroside (Rhodioloside) read alignment due to the presence of chimeric reads and the conversion of unmethylated cytosines to uracils, respectively. We developed TAURUS-MH (Two-step Alignment with Unmapped Reads Using read Splitting for Methyl-HiC), a mapping pipeline for m3C-seq data using a hybrid of ungapped and read splitting alignments (Supplementary Fig. 1a). Sequencing reads were first mapped to an bisulfite converted genome using Bismark calling an ungapped aligner (bowtie1)21, and unmapped reads are split into 3 segments followed by ungapped alignment. We compared the performance of TAURUS-MH to BWA-METH22, which is designed for bisulfite sequencing data alignment using BWA-MEM. This comparison was performed using common Hi-C data with simulated bisulfite conversion. We use the alignment of conventional Hi-C data 23 as our gold standard. When compared with BWA-METH, our pipeline showed 19.43% higher in mappability (86.12% Felypressin Acetate vs. 66.69%, Fig. 2a), 3.64% higher Salidroside (Rhodioloside) in accuracy (97.86% vs. 94.22%, Fig. 2b), and 13.41% higher long-range contacts (42.79% vs. 29.38% from total fragments and 49.68% vs. 44.06% from mapped fragments, Fig. 2c). Open in a separate window Physique 2. Data processing and analysis of m3C-seq sequencing reads.Reads derived from non-bisulfite treated regular Salidroside (Rhodioloside) Hi-C sequencing are converted C to T (read1) and G to A (read2) in silico and aligned using BWA-meth, Bismark (bowtie1), and Bismark (bowtie1) followed by split-read alignment. Alignment of non-converted reads using conventional alignment pipeline is used as a standard (Conventional, Non-converted). For (a-d), the mapping algorithms were applied to a common test dataset (n=1) to make a fair comparison of their performance. (a) Percent of aligned reads as a pair. (b) Alignment accuracy of different alignment strategies compared with conventional Hi-C alignment using converted reads. (c) Fraction of read pairs with cis short-range reads (cis < 1kb), cis long-range interactions (cis > 1kb), and trans interactions (trans) using different alignment strategies. (d) Similar to panel (c), but for 3C-seq (without conversion), bulk m3C-seq (with conversion, from the same sample as bulk 3C-seq), and combined 192 single-nucleus m3C-seq results. (e) Contact maps from chromosome 17 for conventional.