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. 2022 May 30;28(6):1207–1211. doi: 10.1038/s41591-022-01793-4

Fig. 2. Single-cell-derived colony CALR genotyping and neonatal blood spot analysis.

Fig. 2

a, Orthogonal validation of the WGS results at the single-cell level. Somatic variants shared between the twins and somatic variants unique for twin A or twin B were assessed in DNA from fluorescence-activated cell sorting (FACS)-sorted single-HSPC colonies (from twin A) or mini-bulk populations using Sanger sequencing. The samples with presence of the studied variant are shown in red, whereas absence of the variant is shown in blue. Details of the tested variants are shown in Supplementary Table 6. b, Flow cytometry profiles of HSPCs for each of the twins. c, CALR genotyping of single-HSPC-derived colonies, integrated with index sorting data. d, Results of ddPCR analysis for the detection and quantification of JAK2V617F in DNA extracted from neonatal dried blood spots from patients diagnosed with JAK2-mutant myeloproliferative neoplasms as adults. In one out of three patients studied (DBS_4) JAK2V617F was detected in neonatal blood with a fractional abundance of 1.38% (three technical replicates in one experiment, independently validated by nested PCR). FAM channel-positive events on the y axis correspond to JAK2V617F positivity, and HEX channel-positive events on the x axis correspond to JAK2 WT events. JAK2V617F-positive DNA from HEL cells, and JAK2 wild-type DNA from TF-1 cells and a dried blood spot from a patient with systemic mastocytosis (DBS_1), were used as positive and negative controls, respectively. All results were independently validated by a nested PCR method. Bars and error bars show median and 95% confidence interval, respectively. Relevant clinical information for the patients studied is provided in Supplementary Table 7. CMP, common myeloid progenitor (Lin−CD34+CD38+CD123+CD45RA−); GMP, granulocyte macrophage progenitor (Lin−CD34+CD38+CD123+CD45RA+); HSC, Lin−CD34+CD38−CD90+CD45RA−; LMPP, lymphoid-primed multipotent progenitor (Lin−CD34+CD38−CD90−CD45RA+); MEP, megakaryocyte-erythroid progenitor (Lin−CD34+CD38+CD123−CD45RA−); MPP, multipotent progenitor (Lin−CD34+CD38−CD90−CD45RA−); MUT, mutated; Mye, myeloid; PE-Cy7, PE-cyanine7; WT, wild-type.