The analysis of cell-free DNA (cfDNA) in plasma represents a rapidly advancing field in medicine. cfDNA consists predominantly of nucleosome-protected DNA shed into the bloodstream by cells undergoing apoptosis. We performed whole-genome sequencing of plasma DNA and identified two discrete regions at transcription start sites (TSSs) where nucleosome occupancy results in different read depth coverage patterns for expressed and silent genes. By employing machine learning for gene classification, we found that the plasma DNA read depth patterns from healthy donors reflected the expression signature of hematopoietic cells. In patients with cancer having metastatic disease, we were able to classify expressed cancer driver genes in regions with somatic copy number gains with high accuracy. We were able to determine the expressed isoform of genes with several TSSs, as confirmed by RNA-seq analysis of the matching primary tumor. Our analyses provide functional information about cells releasing their DNA into the circulation.
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Integrated supplementary information
Localization of the NDR, which was mapped by analyses of 100 (red) and 1,000 (orange) highly expressed genes in the 104 plasma samples from healthy donors and which was most often observed in a –150 bp to +50 bp window with respect to the TSS (blue, 1,000 most weakly expressed genes).
Support vector machine (SVM) classification based on normalized 2K-TSS and NDR coverage for the 5,000 most highly and least expressed genes. Red and dark blue circles represent genes correctly assigned to the expressed and unexpressed clusters, respectively, whereas light blue and orange circles represent incorrectly assigned genes (as in Fig. 3b).
(a) Correlation between 2K-TSS (left) and NDR (right) coverage and FPKM percentiles. (b) Means and distribution of the 2K-TSS and NDR coverage parameters of genes grouped into deciles. (c) Average FPKM percentile of binned 2K-TSS and NDR coverage parameters.
Supplementary Figure 4 Comparison of copy number profiles of the matching primary tumor with plasma DNA.
The copy number profiles of the matching primary tumors B7 (top) and B13 (bottom) were obtained by whole-genome sequencing with a shallow sequencing depth. Pairwise comparisons of genomic position–mapped profiles revealed high correlations between the copy number profiles (Pearson correlation coefficients = 0.74 (B7) and 0.88 (B13)).
Supplementary Figure 5 Reconstruction of the 12p11.1 nucleosome array with high-coverage sequenced plasma samples.
Assembly of the 12p11.1 nucleosome arrays in plasma samples from B7, B13, controls, and GM12878 for comparison.
Supplementary Figure 6 TSS nucleosome occupancy of unexpressed and housekeeping genes in high-coverage sequenced plasma samples in B7 and B13.
(a,b) Nucleosome occupancy at TSSs of unexpressed genes (fantom.gsc.riken.jp/5/) and housekeeping genes2 had the expected different pattern for B7 (a) and B13 (b).
Histogram of prediction consent in merged control (n=104) data. For the majority of genes, the prediction consent was above 95%; there are only a few genes with a prediction consent below 75%.
Supplementary Figures 1–7 and Supplementary Note. (PDF 1610 kb)
List of the 100 most highly expressed genes based on plasma RNA-seq data provided by Koh et al. but predicted to be unexpressed (n = 12). (XLSX 11 kb)
List of the 1,000 most highly expressed genes based on plasma RNA-seq data provided by Koh et al. but predicted to be unexpressed (n = 245). (XLSX 18 kb)
Subsampling of sequencing data to establish a lower boundary of necessary sequencing coverage. (XLSX 10 kb)
Prediction and FPKM values for genes in focal amplifications having log2 ratio > 1 in breast cancer case B7. (XLSX 19 kb)
Prediction and FPKM values for genes in focal amplifications having log2 ratio > 1 in breast cancer case B13. (XLSX 25 kb)
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Ulz, P., Thallinger, G., Auer, M. et al. Inferring expressed genes by whole-genome sequencing of plasma DNA. Nat Genet 48, 1273–1278 (2016). https://doi.org/10.1038/ng.3648
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