BIOINFORMATICS ANALYSIS OF FULL-LENGTH RNA NANOPORE SEQUENCING AT SINGLE CELL LEVEL TO STUDY TRANSCRIPTIONAL AND MUTATIONAL PATTERNS IN HUMAN LEUKEMIA

Acute myeloid leukemia (AML), an aggressive cancer originating from hematopoietic stem cells, exhibits substantial intra-tumoral heterogeneity impacting disease development, prognosis, and treatment response. However, understanding the interplay between various layers of heterogeneity at the single-cell level remains limited due to technological constraints. To address this, a novel high-throughput multi-omics approach, Single Cell and Molecule sequencing (SCM-seq), was developed. This method integrates droplet-based single-cell RNA sequencing with Nanopore single-molecule sequencing, allowing comprehensive profiling of the tumor ecosystem in AML. SCM-seq was employed to analyze AML samples with SRSF2 spliceosome-factor gene mutations, elucidating the relationship between genetic complexity and transcriptional heterogeneity in malignant and immune compartments. The results confirmed the efficacy of SCM-seq in achieving high-throughput multi-omic profiling at the single-cell level and reconstructing sample complexity. Analyses of expression profiles using reference datasets enabled the identification of malignant and microenvironment compartments within AML samples. Long-read analyses of mutated gene transcripts facilitated the sensitive identification of mutations at the individual cell level. Surprisingly, mutant cells were not confined to the HSC/progenitor-like AML population but also present in differentiated myeloid cells and lymphocytes. Increasing genetic complexity in HSC/progenitor-like AML cells correlated with heightened transcriptional heterogeneity and decreased isoform abundance, indicating a narrowed repertoire of isoforms with elevated genetic complexity. Notably, the SRSF2 mutation consistently correlated with increased isoforms across all lineages. Detailed splicing pattern analyses revealed prevalent exon skipping and intron retention in AML samples. AML cells with the SRSF2 mutation exhibited significantly higher proportions of genes with multiple isoforms or novel transcripts. These findings illuminate the intricate connections between genetic complexity, transcriptional heterogeneity, isoform abundance, and specific gene expressions in diverse lineages of AML, providing valuable insights into disease progression and potential implications for targeted therapeutic strategies.