Senior Scientist/ BA Principal Investigator Moderna Therapeutics Norwood, Massachusetts
We developed and qualified a novel single-cell RNA sequencing (scRNA-seq) assay to profile immune-cell populations in leukocytes from human blood and track an mRNA-LNP therapeutic at single-cell resolution. Leukocytes from pooled blood were cryopreserved and used to evaluate live-cell enrichment, precision, selectivity, specificity, sensitivity, robustness, and short-/long-term stability. Libraries were generated using GEM-X v4 kit (target 20,000 cells) on 10x ChromiumX, QC-checked on TapeStation, quantified and sequenced on Illumina NextSeq2000 or NovaSeq6000 with acceptance thresholds of ≥20,000 reads per cell, ≥200 genes per cell, ≥75 % reference genome alignment, and ≤15 % mitochondrial reads. All criteria were met: viability enrichment to ≥65 %, intra-/inter-assay %CVs ≤50 % for immune subsets, no false-positive therapeutic mRNA reads, sensitivity to 0.2 % positive cells, comparable performance across sequencers, and stability confirmed to six months (monitoring to 12 months). The qualified workflow supports reliable single-cell transcriptomic analysis and bio-distribution assessment of mRNA therapeutics.
Learning Objectives:
Learn about single cell sequencing assay background and applications
Learn to design, execute, and evaluate scRNA-seq assay qualification for clinical applications,
Learn about assay qualification parameters; cell viability, precision, specificity, sensitivity, robustness of the assay across Illumina sequencing platforms, and stability of cryopreserved leukocytes.
