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Abstract Deep targeted sequencing technologies are still not widely used in clinical practice due to the complexity of the methods and their cost. The Molecular Inversion Probes (MIP) technology is cost effective and scalable in the number of targets, however, suffers from low overall performance especially in GC rich regions. In order to improve the MIP performance, we sequenced a large cohort of healthy individuals (n = 4417), with a panel of 616 MIPs, at high depth in duplicates. To improve the previous state-of-the-art statistical model for low variant allele frequency, we selected 4635 potentially positive variants and validated them using amplicon sequencing. Using machine learning prediction tools, we significantly improved precision of 10–56.25% (P < 0.0004) to detect variants with VAF > 0.005. We further developed biochemically modified MIP protocol and improved its turn-around-time to ∼4 h. Our new biochemistry significantly improved uniformity, GC-Rich regions coverage, and enabled 95% on target reads in a large MIP panel of 8349 genomic targets. Overall, we demonstrate an enhancement of the MIP targeted sequencing approach in both detection of low frequency variants and in other key parameters, paving its way to become an ultrafast cost-effective research and clinical diagnostic tool.

Clonal haematopoiesis (CH) arises from the expansion of hematopoietic stem cells (HSCs) carrying leukaemia-associated somatic mutations. CH is linked to pathological immune dysregulation and a greater risk of age-related inflammatory diseases. Yet, how CH mutations impact HSC differentiation into immune effector cells remains understudied. Here, we report a single-cell resolution functional and multi-omic investigation of HSC clonal and differentiation dynamics in individuals with DNMT3A-R882 CH. DNMT3A-R882 reshapes the clonal architecture of haematopoiesis towards an aged phylogenetic structure. Functionally, DNMT3A-R882 HSCs produce decreased monocytic output but more abundant and mature neutrophil progeny compared to WT HSCs in the same individual. Whereas DNMT3A-R882 myeloid progenitors display attenuated inflammatory transcriptional programmes, DNMT3A- R882 mature neutrophils acquire proinflammatory and immunomodulatory features typical of maladaptive immunity and CH co-morbidities. Our findings, validated in humanised mice, identify aberrant DNMT3A-R882 HSC-driven neutropoiesis as a key link between CH, immune dysregulation and risk of inflammatory disease.

Hematopoietic stem and progenitor cells (HSPCs) are intended to deliver life-long, consistent output. However, with age, we observe changes in blood counts and clonal disorders. Better understanding of inter-individual variation in HSPC behavior is needed to understand the transition from health to age-related hematological disorders. Here we study 360K single circulating HSPCs (CD34+) from 99 healthy individuals together with clinical information and clonal hematopoiesis (CH) profiles to characterize population variability in hematopoiesis. Individuals with CH were linked with reduced frequencies of lymphocyte progenitors and higher RDW. We describe a Lamin-A transcriptional signature across the HSPC spectrum and show it is reduced in CH individuals. We define and estimate HSPC composition bias and an age-related increased S-phase gene signature and show how they form a heterogeneous and multifactorial aging trend in the blood. The new comprehensive model of normal HSPC variation will allow the study of stem cell-related disorders. As a proof of concept, we present methodologies for analyzing myeloid malignancies in comparison to our reference atlas. Together, our data and methodologies shed light on age-related changes in blood counts, CH and can be used to study stem cell-related disorders in the future.Competing Interest StatementZS, DL, EM and GY are all employees and shareholders of Ultima Genomics. LS is a shareholder of Sequentify. The remaining authors declare no competing interests