Extended Data Fig. 2: General strategy for the inference of haplotype-specific DNA copy number and chromosome mis-segregation events from single-cell RNA-Seq data.
From: Heritable transcriptional defects from aberrations of nuclear architecture
(a) Two segregation patterns of MN chromosomes (left: 2:2 segregation; right 1:3 segregation) generated by nocodazole-block-and-release and the predicted copy-number outcomes over two generations. MN chromatids (filled magenta) and chromatids of the other haplotype (open magenta) are represented in the same fashion as in Figs. 1 and 2. Under 2:2 segregation, the MN sister cell or the MN nieces (dashed boxes) should display bi-allelic disomic transcription but one of the two MN daughter cells (shaded boxes) should display mono-allelic transcription of the intact haplotype (open magenta) due to deficient replication of the MN chromatid; under 1:3 segregation, the MN sister cell or the MN nieces should display mono-allelic transcription from the intact haplotype (open magenta). The predicted monosomic transcription outcomes are used to identify micronuclear chromosomes and their segregation pattern in each experimental family. (b) and (c) Validation of the transcriptional outcomes of MN (“generation 1”) using an experimental strategy (b) of inducing MN with acentric Chr.5q fragments generated by CRISPR-Cas9 as reported in our recent study25. The data shown in (c) demonstrate the predicted transcriptional outcome when the micronucleus contains only one copy of Chr.5q fragment arm that most closely resembles the segregation patterns generated by nocodazole block-and-release. Two measures of gene transcription are shown: in the left plot, filled and open magenta circles are the normalized allelic expression of the broken and the intact haplotype in 10 Mb bins; on the right are the cumulative TPM (from low to high expression). The MN sister cell shows normal disomic transcription. In the MN cell, monoallelic transcription of the MN haplotype (filled circles) extending from near 64 Mb to the q-terminus indicates silencing of an acentric Chr.5 fragment partitioned into the micronucleus after Cas9-breaks generated at ~64 Mb. Reduced transcription of Chr.5 in the MN cell is also evident from the cumulative TPM plot on the right that shows a reduction in total transcription relative to normal disomic Chr.5. As the cumulative TPM plot is generated for all genes on Chr.5, it does not distinguish chromosome-wide transcriptional reduction from regional loss of transcription. (d) Identification of chromosomes with non-reference transcriptional states (red dots) in MN families (n = 173 cells) based on reference transcription distributions determined from control RPE-1 cells (Extended Data Fig. 1d). Based on the inferred DNA copy-number states of these chromosomes (assuming proportional transcriptional yield and DNA copy number), we further identify chromosomes with mis-segregation patterns consistent with the predicted outcomes in (a). Error bars represent normal range of transcription estimated based on the 5 % and 95 % values in control cells. Red dots represent chromosomes with significant deviations (Bonferroni corrected P <0.05, two-tailed Z-test; for 48 chromosomes including both homologues).
