Supplementary Figure 1 a

Transcription

1 Supplementary Figure 1 a b d c

2 Supplementary Figure 1. Poor sensitivity for mutation detection using established single-cell RNA-sequencing methods. (a) Representative bioanalyzer trace showing expected single-cell library concentration from K562 cells. (b) Detection of BCR-ABL and GAPDH by QPCR in libraries from single K562 cells processed using the standard C1 protocol (b). Values shown are the gene expression levels relative to the limit of detection (LOD), indicated by the dashed horizontal line. The box plot shows median and quartile values and whiskers show outlier values within 1.5 interquartile range of the quartiles. Numbers below each plot show the frequency of cells showing expression above the LOD. (c) Violin plot shows the expression level (log2 RPKM) measured by RNA-sequencing of specific mutation hotspots within genes that are frequently mutated in myeloproliferative diseases (K562, n=15). (d) Detection of BCR-ABL and GAPDH by QPCR in libraries from single K562 cells processed with the C1 protocol with targeted BCR-ABL amplification.

3 Supplementary Figure 2 a b c d

4 Supplementary Figure 2. BCR-ABL tss2 method. (a) Scheme illustrating the workflow for single-cell detection of BCR-ABL with parallel whole transcriptome analysis. Single-cells are sorted into individual wells of a 96- well plate containing lysis buffer. BCR-ABL specific primers are multiplexed during both reverse transcription (RT) and PCR amplification steps to improve detection of BCR-ABL transcripts. The quality, size and concentration of the resulting cdna libraries are assessed by Agilent High Sensitivity bioanalyzer chips. Single-cell derived cdna libraries are then genotyped for BCR-ABL expression relatively to GAPDH housekeeping gene by QPCR and each library is assigned with a score of BCR-ABL positive or negative. cdna libraries are then indexed with Illumina Nextera XT kit and 40 to 50 singlecells are pooled together to be sequenced on a single lane of a HiSeq2000. (b) Table summarizing the BCR-ABL primers used for BCR-ABL tss2. Primer set #2 contains the Smart-seq2 ISPCR oligo sequences in the 5 end of each oligo. (c) Table illustrating the optimization of the protocol for BCR-ABL tss2 in single K562 cells. In addition to standard Smart-seq2 (condition 0), different combinations for tss2 multiplexing primers sets #1 and #2 during RT and PCR amplification are shown (conditions 1-6). For each of the combinations tested, primer sets and their relative concentration in the RT and PCR steps are shown. For each of the conditions used (0-6) we show the frequencies of BCR-ABL detection by QPCR in single K562 cells, the average CT value at which BCR-ABL gene was detected by QPCR and (d) a representative bioanalyzer trace of the cdna libraries obtained. Absence of BCR-ABLspecific primers in the RT step (conditions 0,1,2) led to poor detection of the fusion transcript in single K562 (29%, 0% and 33%, respectively). The presence of primer set #1 in both RT and PCR (condition 3), brought the frequency of BCR-ABL detection up to 100% but with QPCR CT values above 30. When primer set #2 was used in both RT and PCR stages (conditions 4 and 5), the presence of ISPCR sequences during the RT step caused the formation of concatamers at an extent that was proportional to their concentration in the reaction mix. Optimal BCR-ABL tss2 conditions (condition 6), were with primer set #1 during RT and primer set #2 during PCR

5 step, allowing 100% of BCR-ABL detection in single K562 with BCR-ABL detection by QPCR at CT values below 30 and with no concatamer formation in the cdna libraries.

6 Supplementary Figure 3

7 Supplementary Figure 3. Unbiased libraries are generated using the BCR- ABL tss2 method. Pearson s hierarchical clustering of RNA-sequencing results from single K562 cells processed by Smart-seq2 (blue n=38) or by BCR-ABL tss2 (red n=38).

8 Supplementary Figure 4 a b c Ensemble of single cells (n=42) d RPKM (Mean Log2) Bulk (100 cells) ERCC concentration (Log2) e

9 Supplementary Figure 4. BCR-ABL tss2 detects transcriptional heterogeneity in normal human HSCs. (a) Representative bioanalyzer traces of cdna libraries generated from single human Lin - CD34 + CD38 - HSCs using the BCR-ABL tss2 protocol. (b) Histogram showing the numbers of genes detected with RPKM 1 in HSCs (n=232) from 5 normal donors. (c) Correlation between the merged data from 42 single-cells from a normal donor ( ensemble ) and the bulk (100 cells) RNA-sequencing measurement of gene expression from the same donor. The ensemble was created by computationally pooling all the reads obtained from the 42 single Lin - CD34 + CD38 - cells the same normal BM donor. (d) Correlation between measured expression level by single-cell BCR-ABL tss2 RNA-sequencing (yaxis) and actual numbers of artificial ERCC RNA genes spiked-in per reaction (x-axis). (e) Pearson correlation coefficient of single-cell BCR-ABL tss2 RNAsequencing libraries showing increased heterogeneity of gene expression between human HSCs than between K562 cells.

10 Supplementary Figure 5 a b 2.03E-02 BCR-ABL- SCs 8.82E E E-01 BCR-ABL+ SCs Normal HSC 7.50E E E E-01 c BCR-ABL- SCs BCR-ABL+ SCs Normal HSC

11 Supplementary Figure 5. Comparison of gene expression by BCR-ABL tss2 RNA-sequencing and QPCR. We selected 12 genes of potential biologic interest showing over 10-fold differential expression for further validation by QPCR, along with 12 non-differentially expressed controls. (a) Correlation between level of expression measured by RNA-sequencing and Q-PCR for 24 selected genes. Differentially expressed genes between BCR-ABL + and BCR- ABL SCs (red dots, n=12) were selected by setting a fold change cutoff >10 and among those 12 genes of potential biologic interest were picked. Nondifferentially expressed control genes (grey dots, n=12) were selected as housekeeping genes or relevant genes for the cell type analyzed (b, c) Beeswarm plots for selected control genes not differentially expressed between BCR-ABL + and BCR-ABL SCs by both RNA-sequencing (b) and QPCR (c) data. Numbers of cells analyzed and numbers of cells showing amplification for the selected gene are shown below the plot. Nonparametric Wilcoxon test p-values are shown on top of each bar graph. Fisher s exact test p-values are shown below the graph. The average gene expression levels are indicated by red squares, the median and quartiles of gene expression levels are represented by the boxes. The dashed lines represent the LOD.

12 Lineage CD38 CD90 CD123 CP-CML CML on TKI Supplementary Figure 6 Live Lineage- Lin-CD34+CD38- Lin-CD34+CD38+ 49, Normal Bone Marrow FSC-A CD34 CD45RA CD45RA

13 Supplementary Figure 6. The composition of the Lin - CD34 + CD38 - HSC compartment is relatively preserved in CML. Representative FACS profile and gating strategy of BM cells from the analyzed normal donors (n=5) and CP- CML patients at diagnosis (n=18) and during TKI therapy (patients n=16; timepoints on TKI n=20). Figures show the mean percentage of parent gate for stem and progenitor populations across all analyzed donors/patients. The parent gate is indicated on top of the FACS plots.

14 Supplementary Figure 7 a b c d e f h g

15 Supplementary Figure 7. Overexpression of proliferation associated genes in BCR-ABL + CML-SCs. (a) Histogram showing the numbers of genes detected with RPKM 1 in 854 single-cells from 18 CML patients at diagnosis (mean=3,591). (b, c) Read depth (b) and mapped reads (c) per cell of normal-hscs (n=232), BCR-ABL SCs (n=377) and BCR-ABL + SCs (n=477) (d). Beeswarm plot showing comparable gene expression level of B2M in normal-hscs (n=232), BCR-ABL SCs (n=377) and BCR-ABL + SCs (n=477). (e) Number of detected genes in normal-hscs, BCR-ABL SCs and BCR- ABL + SCs. (f, g) Gene-set enrichment analysis comparing expression of proliferation- (f) and quiescence- (g) associated gene expression signatures in BCR-ABL SCs in comparison with normal-hscs. (h) The box plot illustrates the frequency of co-expression of G2/M associated genes among normal-hscs (black, n=232), BCR-ABL SCs (blue, n=377) and BCR-ABL + SCs (red, n=477). The frequency of co-expression of G2/M transition genes was higher in BCR-ABL + SCs when compared to either BCR-ABL SCs or normal-hscs (p=1.52e-07 and p=2.67e-06, respectively).

16 Supplementary Figure 8 a 5.77E E E E-09 Normal HSC BCR-ABL- SCs BCR-ABL+ SCs 3.35E E E E E E E E E E E-09 b 3.78E E E E E E E E E E E E E E E E E E E E E E E E E-21 c A: C: E: B: D: Normal HSC BCR-ABL- SCs BCR-ABL+ SCs

17 Supplementary Figure 8. Single-cell analysis enhances resolution for detection of aberrant gene expression in BCR-ABL + and BCR-ABL SCs. Beeswarm plots for selected differentially expressed genes between normal- HSCs, BCR-ABL and BCR-ABL + SCs showing log2(rpkm) for differentially expressed genes previously related to CML pathogenesis (a) and uniquely identified by this study (b). Numbers of cells analyzed and numbers of cells showing expression of the selected gene are shown below the plot. Nonparametric Wilcoxon test p-values (normal-hscs vs BCR-ABL + SCs) are shown on top of each bar graph. Fisher s exact test p-values (normal-hscs vs BCR-ABL + SCs) are shown below the graph. The average gene expression levels are indicated by red squares, the median and quartiles of gene expression levels are represented by the boxes. (c) Venn diagram showing differentially expressed genes detected using data analyzed as an ensemble of single-cells (in silico bulk) in comparison with differentially expressed genes detected using single-cell analysis (232 normal-hscs; n=5 subjects versus 477 BCR-ABL + SCs and 377 BCR-ABL SCs CP-CML; n=18 patients). Five groups of genes are highlighted in the Venn diagram: A) genes differentially expressed only in single BCR-ABL + SCs vs single normal-hscs, B) genes differentially expressed only in single BCR-ABL SCs vs single normal-hscs, C) genes differentially expressed in both single BCR-ABL + and single BCR- ABL SCs vs single normal-hscs, D) genes differentially expressed between CML SCs and normal-hscs only in bulk analysis (typically very low level expressed genes), E) genes differentially expressed in both single-cell analysis and in silico bulk analysis. For each group an example gene is shown.

18 Supplementary Figure 9 a b

19 Supplementary Figure 9. Differentially expressed genes between normal- HSCs and BCR-ABL + SCs in individual CML patients. (a) Heatmap showing degree of up- or down-regulation for 116 differentially expressed genes between normal-hscs and BCR-ABL + SCs, according to the indicated log2(fc) scale. Each column represents an individual diagnostic patient. (b) The same genes as for panel (a), now showing whether differential expression was significant (P< 0.05 Fisher s test) in each patient.

20 Supplementary Figure 10

21 Supplementary Figure 10. Single-cell whole transcriptome analysis of BCR- ABL + SCs confirms aberrant gene-expression derived from previous CML stem/progenitor cell datasets. GSEA performed using selected gene-sets previously described in CML related literature for 1) Normal-HSCs (n=6) vs total (BCR-ABL and BCR-ABL + SCs combined) CML-SCs (n=18) as an in silico bulk analysis; 2) Single-cell analysis of normal-hscs from 5 normal donors (n=232) vs BCR-ABL SCs from 18 patients (n=377); 3) Single-cell analysis of normal-hscs (n=232) vs BCR-ABL + SCs (n=477) from 18 CML patients; 4) Single-cell analysis of BCR-ABL SCs (n=377) vs BCR-ABL + SCs (n=477) from 18 CML patients. A false discovery rate (FDR) cut-off of 0.25 was used.

22 Supplementary Figure 11 p=0.006 p< p< p<0.0001

23 Supplementary Figure 11. Measurement of time required by CML-SCs to undergo first cell division upon stimulation with TNF and TGF. The dot plot shows the number of single Lin - CD34 + CD38 - cells having undergone the first cell division after 96 hours in culture in standard cytokine condition and in the presence of TNF (20ng/ml) or TGF (20ng/ml). The result is shown for Lin - CD34 + CD38 - isolated from the BM of healthy controls (n=2) or CP-CML (n=4). The number is expressed as divided cells per teraski plate (60 wells). Chi-square p-values are shown in the box plot. Whiskers represent minimum and maximum values, the line represents the mean.

24 Supplementary Figure 12 a b OX1570 CML656 CML22 OX1902 CML2286

25 Supplementary Figure 12. Single-cell analysis reveals distinct molecular signatures of quiescent BCR-ABL + CML-SCs persisting during TKI therapy. (a,b) tsne visualization of normal-hscs and BCR-ABL + SCs at diagnosis and remission, generated using top 500 genes (Supplementary Table 7). In the background, normal-hscs (dim grey circles, n=232; 5 subjects) and BCR- ABL + SCs from all patients at diagnosis (dim grey triangles, n=477; 18 patients) and remission (dim red circles, n=245, 16 patients) are shown. In each plot single BCR-ABL + SCs from individual patients are highlighted by bright color-coding specific for each time-point. Panel (a) shows patients with relative enrichment for group-a remission cells during TKI therapy who subsequently achieved MMR and (b) shows two patients with predominantly group-b remission CML-SCs; the upper panel shows a patient who failed to achieve therapeutic Imatinib levels and the lower panel shows a patient who had temporarily interrupted TKI therapy at the time the sample was taken.

26 Supplementary Figure 13

27 Supplementary Figure 13. Single-cell analysis reveals distinct molecular signatures of BCR-ABL + CML-SCs during TKI therapy. GSEA heatmaps generated using normal-hscs (n=232, 5 normal subjects) vs group-a BCR- ABL + SCs at remission (n=122, 16 patients) vs BCR-ABL SCs at remission (n=420, 16 patients).

28 Supplementary Figure 14 a b

29 Supplementary Figure 14. Single-cell analysis reveals distinct molecular signatures of BCR-ABL + SCs at BC. (a) tsne visualization as shown in Fig.6a but with cells from patient 1266 highlighted. The color code highlights cells from paired pre-bc sample (taken at diagnosis when patient was in CP; green diamonds, n=53), and BC sample (taken at the time of myeloid blast crisis transformation; bright purple squares, n=63) of patient In the background are shown normal-hscs from 5 donors (dim grey circles; n=232), BCR-ABL + SCs from 18 CP-CML patients (dim red triangles; n=477) BCR- ABL + SCs from other blast crisis patients (OX1931 pre-bc, dim orange diamonds, n=132; OX1931 BC, dim light-blue squares, n=85; CML1203 BC, dim pink squares, n=7) and K562 cells (dim brown circles, n=53). (b) Heatmap showing the QPCR validation of selected genes in single BCR- ABL + SCs from two typical CP-CML patients at diagnosis in single BCR- ABL + SCs (OX664 n=32; OX2038 n=27) and BCR-ABL SCs (OX664 n=12; OX2038 n=17), and from the pre-bc sample from patient OX1931 (n=125).

30 CD38 CD90 CD123 Supplementary Figure 15 Live Lineage - Lin-CD34+CD38- Lin-CD34+CD Pre-BC CML BC CML Pre-BC OX BC OX Lineage BC CML FSC-A CD34 CD45RA CD45RA

31 Supplementary Figure 15. FACS profiles and gating strategy of BM cells from BC patients. The figure shows the BM composition of pre-bc samples (n=2; OX1931 and CML1266), lymphoid BC samples (n=2; OX1931 and CML1203) and myeloid BC samples (n=1; CML1266) analyzed. Numbers are percentages of parent gate for each gated stem and progenitor population. The parent gate is indicated on top of the FACS plots.

32 Supplementary Figure 16 a b Pre-BC OX1931 BC OX1931 c

33 Supplementary Figure 16. Tracking clonal evolution in single CML SCs. (a) Exome sequencing analysis identifies RUNX1 c.g521a mutation in both pre- BC and BC single SCs from patient OX1931. (b) The dot plot shows the index sort results corresponding to individual BCR-ABL + SCs from pre-bc sample. The color indicates if cells were in the CML CP-cluster (blue) or in the BC cluster (red). The values are expressed as mean fluorescent intensity (MFI) for CD90 and CD45RA antigens (y and x axis, respectively). (c) Heatmap showing QPCR validation in single BCR-ABL + SCs from pre-bc OX1931. The heatmap shows a panel of genes selected as differentially expressed between CP-CML cluster and BC-CML cluster (as shown in Fig. 6a), together with a SNP assays specific for RUNX1 WT or RUNX1 c.g521a (RUNX1 mut).

34 Supplementary Tables Supplementary Table 1. Patient demographics and characteristics Supplementary Table 2. Differentially expressed genes between normal HSCs, BCR-ABL + and BCR-ABL SCs from CP-CML patients at diagnosis. (a) References for some differentially expressed genes between BCR-ABL + CML SCs and normal HSCs. Both genes that have been previously implicated in CML and novel candidates identified by this study have been included; (b) Differentially expressed genes between normal HSCs and BCR-ABL + SCs from CP-CML patients at diagnosis; (c) Differentially expressed genes between normal HSCs and BCR-ABL SCs from CP-CML patients at diagnosis; (d) Differentially expressed genes between BCR-ABL SCs and BCR-ABL + SCs from CP-CML patients at diagnosis. Supplementary Table 3. Gene-sets from previous studies on CML stem and progenitor cells Supplementary Table 4. Results from GSEA comparing normal HSCs to BCR-ABL + SCs and BCR-ABL SCs from CP-CML patients at diagnosis and using gene-sets from previous studies on CML stem and progenitor cells (Supplementary Table 3). (a) GSEA of BCR-ABL + SCs and BCR-ABL SCs from CP-CML patients at diagnosis; (b) GSEA of BCR-ABL + SCs from CP- CML patients at diagnosis against normal HSCs; (c) GSEA of BCR-ABL SCs from CP-CML patients at diagnosis against normal HSCs. Supplementary Table 5. Results from GSEA comparing normal HSCs to BCR-ABL + SCs and BCR-ABL SCs from CP-CML patients at diagnosis and using HALLMARK gene-sets. (a) HALLMARK gene-sets; (b) GSEA on HALLMARK gene-sets of BCR-ABL + SCs and BCR-ABL SCs from CP-CML patients at diagnosis; (c) GSEA on HALLMARK gene-sets of BCR-ABL + SCs from CP-CML patients at diagnosis against normal HSCs; (d) GSEA on

35 HALLMARK gene-sets of BCR-ABL SCs from CP-CML patients at diagnosis against normal HSCs. Supplementary Table 6. Results from GSEA comparing diagnostic samples from good and poor responder CML patients. (a) GSEA on HALLMARK genesets comparing BCR-ABL SCs from good responders to BCR-ABL SCs from poor responders; (b) GSEA on HALLMARK gene-sets comparing BCR- ABL + SCs from good responders to BCR-ABL + SCs from poor responders; (c) GSEA on CML-related gene-sets (Supplementary Table 3) comparing BCR- ABL SCs from good responders to BCR-ABL - SCs from poor responders; (d) GSEA on CML-related gene-sets (Supplementary Table 3) comparing BCR- ABL + SCs from good responders to BCR-ABL + SCs from poor responders. Supplementary Table 7. Top 500 informative genes for distinguishing normal-hscs from BCR-ABL + SCs at diagnosis and during remission. Supplementary Table 8. Results from GSEA on HALLMARK gene-sets comparing remission group-a BCR-ABL + SCs to remission group-b BCR- ABL + SCs. Supplementary Table 9. Differentially expressed genes between normal HSCs, BCR-ABL+ SCs from diagnosis, remission group-a and remission group-b. (a) Differentially expressed genes between normal HSCs and BCR- ABL + SCs remission group-a; (b) differentially expressed genes between BCR-ABL + SCs remission group-a versus BCR-ABL + SCs remission group-b; (c) differentially expressed genes between BCR-ABL - SCs from CP-CML patients at diagnosis and BCR-ABL + SCs remission group-a; (d) differentially expressed genes between BCR-ABL + SCs from CP-CML patients at diagnosis and BCR-ABL + SCs remission group-a. Supplementary Table 10. Results from GSEA comparing remission group-a BCR-ABL + SCs to normal HSCs and remission BCR-ABL SCs. (a) GSEA on

36 HALLMARK gene-sets comparing remission group-a BCR-ABL + SCs to normal-hscs; (b) GSEA on CML-related gene-sets comparing remission group-a BCR-ABL + SCs to normal-hscs; (c) GSEA on HALLMARK gene-sets comparing remission group-a BCR-ABL + SCs to remission BCR-ABL SCs; (d) GSEA on CML-related gene-sets comparing remission group-a BCR- ABL + SCs to remission BCR-ABL SCs. Supplementary Table 11. Differentially expressed genes between single BCR-ABL + SCs falling in CP-CML cluster and BCR-ABL + SCs falling in BC- CML cluster (related to Fig. 6a).

37 Supplementary Table 12. Anti-human FACS Antibodies. Antigen Clone Conjugate Company CD34 8G12 APC Biolegend CD38 HIT2 PETXR Life Technologies CD90 5E10 PE Biolegend CD45RA MEM56 FITC Life Technologies CD123 6H6 PECy7 Biolegend CD2 RPA-2.10 PECy5 Biolegend CD3 HIT3a PECy5 Biolegend CD4 RPA-T4 PECy5 Biolegend CD7 CD7-6B7 PECy5 Biolegend CD8a RPA-T8 PECy5 Biolegend CD10 HI10a PECy5 Biolegend CD11b ICRF44 PECy5 Biolegend CD14 RMO52 PECy5 Biolegend CD19 HIB19 PECy5 Biolegend CD20 2H7 PECy5 Biolegend CD56 B159 PECy5 BD CD235ab HIR2 PECy5 Biolegend

38 Supplementary Table 13. Reagents mixes for generation of single-cell cdna libraries using BCR-ABL tss2 protocol. Lysis Mix reagents Reagent Volume for 1 cell (μl) 0.4% Triton X + RNAse Inhibitor (1:20) 2 dntps (10 mm) 1 Oligo dt (10 μm) 1 ERCC (pre-diluted 1:400,000) 0.1 TOTAL 4 (4.1 with ERCC) RT mix reagents Reagent Volume for 1 cell (μl) Superscript II first strand buffer (5x) 2 DTT (100 mm) 0.5 Betaine (5 M) 2 MgCl2 (1 M) 0.1 RNAse Inhibitor (40 U/μL) 0.25 TSO (100 μm) 0.1 BCR-ABL Primer set #1 F+R (200 μm) 0.07 Superscript II (200 U/μL) 0.25 Water 0.33 TOTAL 5.6 PCR mix reagents Reagent Volume for 1 cell (μl) KAPA Hifi HS Ready Mix (2x) 12.5 ISPCR oligo (10 μm) BCR-ABL Primer set#2 F+R (20 μm) 0.07 Water TOTAL 15

39 Supplementary Table 14. Modified C1 PCR MIX for BCR-ABL targeted amplification. Reagent Volume (μl) PCR Water (Advantage 2 Kit) X Advantage 2 PCR Buffer 10 (Advantage 2 Kit) 50X dntp Mix (Advantage 2 Kit) 4 IS PCR primer (Clontech SMARTer) 4 50X Advantage 2 Polymerase Mix 4 (Advantage 2 Kit) C1 Loading Reagent (Fluidigm) 4.5 BCR-ABL Taqman assay pre-diluted 4 1:22 (Hs _ft)

40 Supplementary Table 15. Taqman assays used for Fluidigm Dynamic Array. Gene Symbol ABL1 ATG3 B2M BCL2 BCR BCR-ABL BLNK CD33 CD34 CD79A CD79B CD164 CDK6 CKLF CLU CSF1R CTNNB1 CXCR4 DNTT FCER1A GAPDH GAS2 GOLGA8A HPRT HSP90A1 IFITM1 IGF1R IGJ ITGA6 MEIS1 MLLT3 MMRN1 MPL MZB1 PTRF RGS2 RXFP1 SAT1 SELL SELP SOD2 TESPA1 VWF Taqman Assay ID Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _ft Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _g1 Hs _m1 Hs _m1 Hs _s1 Hs _m1 Hs _m1 Hs _m1 Hs _s1 Hs _m1 Hs _m1 Hs _g1 Hs _m1 Hs _m1 Hs _m1 Hs _g1 Hs _s1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _g1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1