Extended Data Fig. 2: Detailed analyses of scRepli-seq profiles of 1-, 2-, 4-cell embryos.

(a,b) (left) NGS tag density plots of mid-S scRepli-seq data (three individual cells) from 1-cell (a) and 2-cell embryos (b) with >30 M total NGS reads per cell. Density was calculated using sliding windows of 200 kb at 40-kb intervals, 80 kb at 16-kb intervals, or 40 kb at 8-kb intervals. In all cases, bimodality is not visible. (right) scRepli-seq NGS read density plots of representative mid-S single-cell data from 1-cell (a) and 2-cell embryos (b) with 78.3 M and 45.9 M reads, respectively, with the indicated bin size settings. Binarization calling outputs are also shown, which revealed the lack of bimodal distribution regardless of the binarization modes chosen. The dotted line shows the lower read count limit of 20 that we set for the unreplicated bins (see Supplementary Note 1). (c) Binarized mid-S scRepli-seq profiles derived from mESCs, 1-cell embryos, and 2-cell embryos with high read counts (18.4–19.9, 30.7–78.3, 34.5–46.6 M reads respectively). The indicated bin size settings and binarization modes were chosen. Unlike mESCs, 1- and 2-cell embryos lacked bin-to-bin variation, showing either seemingly totally ‘unreplicated’ or totally ‘replicated’ profiles depending on the binarization mode (1-somy or 2-somy; see Methods). (d) NGS tag density plots of representative 1/2/4-cell and mESC scRepli-seq profiles during cell cycle at G1 and S (S1–S4), which are indicated in (e). Tag densities were calculated for 200-kb sliding windows at 40-kb intervals. For (d–k), we used scRepli-seq data with 4–6 M total reads (see Supplementary Note 1). (e) Binarized scRepli-seq profiles of 1/2/4-cell B6MSM embryos on chr15 (80-kb bins). For binarization by AneuFinder⁵⁹, 2-somy mode was applied to 1-, 2- and mid/late 4-cell blastomeres, and 1-somy mode to early 4-cell blastomeres (see Methods for more details). 1- and 2-cell scRepli-seq profiles are ordered based on their sampling order, as we were unable to calculate their percentage replication score. Asterisks, data reflecting gradual and uniform replication (see Fig. 1d legend for details). (f) MAD score distribution of 1/2/4-cell blastomeres analysed by scRepli-seq with 40-kb, 80-kb, and 500-kb bins. MAD scores were constant during S-phase in 1- and 2-cell embryos, in sharp contrast to 4-cell embryos, which exhibited inverted V-shape patterns with the MAD score peaking at mid-S regardless of bin size settings (40, 80, and 500-kb bins). (g) Haplotype-resolved MAD score distribution in 1- and 2-cell embryos. Relatively high scores were detected on the 1-cell paternal genome, although they were lower than somatic cells. (h) Binarized 1-cell S-phase scRepli-seq profiles of the MSM (paternal) chromosome, along with BrdU-IP population RT profiles of mESCs. The averaged scRepli-seq profile (Ave. scRT) was generated by calculating the mean of scRepli-seq data derived from S-phase cells on the MSM (paternal) chromosome. The sperm A/B compartment profile (Hi-C PC1) was calculated from published Hi-C data⁶⁸. RT class definition is based on Dileep et al.²⁰ (CE, constitutively early; CL, constitutively late; D, developmentally regulated). Asterisks, data reflecting gradual and uniform replication (see Fig. 1d legend for details). (i) Percentages of heterogeneously late RT (Ave. scRT<0.75) regions (chrX excluded) on the paternal and maternal genome in 1- and 2-cell embryos. They were observed specifically on the 1-cell paternal genome. (j) RT class distribution of all genomic bins and heterogeneously late RT regions on the 1-cell paternal genome (chrX excluded from analysis). The majority of the heterogeneously late RT regions were classified as constitutively late-replicating (CL). (k) A/B compartment (Hi-C PC1) category distribution of all genomic bins and heterogeneously late RT regions on the 1-cell paternal genome. A1 (strongest A), A2, B2, and B1 (strongest B) each contain 25 % of all PC1 values (200-kb bins) based on a sperm Hi-C data⁶⁸. The majority of the heterogeneously late RT regions were classified as B compartment (heterochromatin), B1 and B2.