Journal: Nature

Article Title: Genotyping of Transcriptomes links somatic mutations and cell identity

doi: 10.1038/s41586-019-1367-0

Figure Lengend Snippet: a, Schematic of GoT workflow. b, Species-mixing study with mutant CALR murine cells and wildtype CALR human cells. 10x reads from singlet cells map to human or murine genome (left). Murine vs. human genome alignment of 10x data (y-axis) and GoT data (x-axis, right, n = 1,259 cells). c, FACS of CD34 + cells (left) and UMI per cell (right) for CALR transcript (blue shade) or targeted locus (pink shade) from representative ET01 (n = 6811 cells) of 10 independent experiments ( , for replicates). d, t-SNE projection of CD34 + cells from ET patients with cluster assignment and e, genotyping data. f, Normalized mutant cell frequency . Bars show aggregate analysis of ET01-ET05 with mean±SD of 100 downsampling iterations to 1 genotyping UMI; points represent mean of n = 100 downsampling iterations for each sample. g, Normalized mutant cell frequency; mean±SD of n = 100 downsampling iterations (Wilcoxon rank-sum test, two-sided). h, t-SNE projection of ET CD34 + cells with pseudotime (left) and density plot of wildtype and mutant cells (right). i, Pseudotime in wildtype vs. mutant cells. P -value from likelihood ratio test of LMM with/without mutation status . j, Bulk VAF of CALR mutation in FACS-sorted cells from ET patients by droplet digital (dd) PCR. HSPC, hematopoietic stem progenitor cells; IMP, immature myeloid progenitors; NP, neutrophil progenitors; M/D, monocyte-dendritic cell progenitors; E/B/M, eosinophil, basophil, mast cell progenitors; MEP, megakaryocytic-erythroid progenitors; MkP, megakaryocytic progenitors; EP, erythroid progenitors; PreB, precursor B-cells; cc, cell cycle; WT, wildtype. MUT, mutant; NA, not assignable. In all figures, box plots represent the median, bottom and upper quartiles, whiskers correspond to 1.5x the interquartile range; violin plots depict kernel density estimates to show the density distribution.

Article Snippet: Peripheral blood from three ET patients with mutations in CALR underwent Ficoll density gradient separation, immunomagnetic selection for CD34 + cells (Miltenyi Biotech), and fluorescence-activated cell sorting (FACS) (Influx, Becton-Dickinson) using PeCy7-labeled CD34, clone 561 (lot# B257238, BioLegend), APC-labeled CD38, clone HIT2 (lot #B247250, BioLegend) and FITC-labeled CD10, clone HI10a (lot# B254556, BioLegend) antibodies were used to isolate CD34 + CD38 − , CD34 + CD38 + , and CD34 + CD10 + cell compartments.

Techniques: Mutagenesis

Journal: Nature

Article Title: Genotyping of Transcriptomes links somatic mutations and cell identity

doi: 10.1038/s41586-019-1367-0

Figure Lengend Snippet: a, Percentage of cells by number of UMIs with CALR mutation locus capture in standard 10x data (left) and GoT data (right) (see Extended Data Fig. 3c for cell number in each sample). b, Number of UMIs per cell of CALR transcript from standard 10x data (left, blue shade) or targeted CALR locus from standard 10x or GoT (pink shade, see Extended Data Fig. 3c for cell number in each sample). c, Summary of clinical, pathologic, and GoT data from patients with CALR -mutated myeloproliferative neoplasms. BM, bone marrow; PB, peripheral blood. d, Number of genes per cell (left) and number of UMIs per cell (right) from published standard 10x data of healthy control CD34 + cells and 10x data from 3’ v2 chemistry of CD34 + cells from patient samples that underwent concurrent GoT, after random down-sampling of the reads from each sample to 50 million reads x3 iterations, showing that extra cycle of PCR and portioning a small aliquot from the 10x cDNA library for GoT using 3’ v2 chemistry does not compromise scRNA-seq data.

Article Snippet: Peripheral blood from three ET patients with mutations in CALR underwent Ficoll density gradient separation, immunomagnetic selection for CD34 + cells (Miltenyi Biotech), and fluorescence-activated cell sorting (FACS) (Influx, Becton-Dickinson) using PeCy7-labeled CD34, clone 561 (lot# B257238, BioLegend), APC-labeled CD38, clone HIT2 (lot #B247250, BioLegend) and FITC-labeled CD10, clone HI10a (lot# B254556, BioLegend) antibodies were used to isolate CD34 + CD38 − , CD34 + CD38 + , and CD34 + CD10 + cell compartments.

Techniques: Mutagenesis, Control, Sampling, cDNA Library Assay

Journal: Nature

Article Title: Genotyping of Transcriptomes links somatic mutations and cell identity

doi: 10.1038/s41586-019-1367-0

Figure Lengend Snippet: a, Wildtype and mutant cell frequency in HSPCs vs. MkPs with variable minimum genotyping UMI thresholds (Fisher’s exact test, two-sided, see for sample size). b, Pseudotime comparison between wildtype and mutant cells with increasing number of thresholds for targeted genotyping UMI (t-test, two-sided, ). c, Pseudotime comparison between mutant and wildtype cells with UMI threshold of 1 (Extended Data Figure 5b) with statistical test using a generalized linear model including mutation status and total number of amplicon UMIs per cell. d, Across 100 iterations, the genotyping amplicon UMIs were downsampled to one per cell and mutant cell frequency was determined for MkPs or precursor B-cells (PreB). This frequency was then divided by the total mutant cell frequency across all progenitor subsets for each of the 100 iterations. Mean ± standard deviation (SD) after n = 100 down-sampling iterations (Wilcoxon rank-sum test, two sided). ET samples with at least 20 cells in each cluster were analyzed. e, Variant allele fraction of CALR mutation in CD34 + , CD38 − (left), CD34 + , CD38 + (middle) and CD34 + , CD10 + (right) FACS-sorted peripheral blood cells from patients with ET determined by droplet digital (dd) PCR.

Article Snippet: Peripheral blood from three ET patients with mutations in CALR underwent Ficoll density gradient separation, immunomagnetic selection for CD34 + cells (Miltenyi Biotech), and fluorescence-activated cell sorting (FACS) (Influx, Becton-Dickinson) using PeCy7-labeled CD34, clone 561 (lot# B257238, BioLegend), APC-labeled CD38, clone HIT2 (lot #B247250, BioLegend) and FITC-labeled CD10, clone HI10a (lot# B254556, BioLegend) antibodies were used to isolate CD34 + CD38 − , CD34 + CD38 + , and CD34 + CD10 + cell compartments.

Techniques: Mutagenesis, Comparison, Amplification, Standard Deviation, Sampling, Variant Assay

Journal: Nature

Article Title: Genotyping of Transcriptomes links somatic mutations and cell identity

doi: 10.1038/s41586-019-1367-0

Figure Lengend Snippet: a, Single-cell cloning assay of peripheral blood cells from MF05 patient (see ). b, Rate of targeted locus capture (%) as a function of gene expression and distance of targeted locus from the transcript ends. c, Distance of mutation locus from transcript ends for pan-cancer drivers and their frequencies based on the number of times reported in the Catalogue of Somatic Mutations in Cancer (COSMIC) database. Mutations are annotated as oncogenes, tumor suppressor genes, or passengers (as defined in Vogelstein et al . 2013 and Bailey et al . 2018 ). Relative density of each subclass of mutations from the closer end (i.e. 3’ or 5’) is shown in the upper panel. d, Schematic of analysis of Oxford Nanopore Technology (ONT) sequencing reads. e, Frequency of SF3B1 mutant and wildtype reads of linear GoT amplicon library sequenced with ONT. f, Analysis of SF3B1 amplicon reads sequenced by Oxford Nanopore Technology (ONT) for inter-transcript PCR recombination by mapping 50 bps at the opposite end of the targeted locus showing only 2.2% of fragments that reflect inter-transcript recombination. g, Pairwise difference of read lengths for duplicate reads (i.e. reads with the same CB+UMI barcodes) of SF3B1 amplicon library sequenced with ONT, showing consistent read length of duplicates supporting a low rate of intra-transcript PCR recombination. h, Comparison of genotype assignment for CALR in patient sample MF01 between linear GoT and circularization GoT after downsampling reads to 300K with 10 iterations (n = 320 cells). i, Comparison of CALR -mutant UMI fraction per cell in patient sample MF01 between linear GoT and circularization GoT after downsampling reads to 300K with 10 iterations (n = 320 cells, Pearson’s correlation, F-test).

Article Snippet: Peripheral blood from three ET patients with mutations in CALR underwent Ficoll density gradient separation, immunomagnetic selection for CD34 + cells (Miltenyi Biotech), and fluorescence-activated cell sorting (FACS) (Influx, Becton-Dickinson) using PeCy7-labeled CD34, clone 561 (lot# B257238, BioLegend), APC-labeled CD38, clone HIT2 (lot #B247250, BioLegend) and FITC-labeled CD10, clone HI10a (lot# B254556, BioLegend) antibodies were used to isolate CD34 + CD38 − , CD34 + CD38 + , and CD34 + CD10 + cell compartments.

Techniques: Cloning, Gene Expression, Mutagenesis, Sequencing, Amplification, Comparison