Performance of the 10X Genomics Flex Single-Cell Sequencing Assay and its Application to Overcome Challenges in Clinical Trial Samples

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Abstract Background Single-cell RNA sequencing (scRNA-seq) has become essential for understanding disease biology, yet its application in clinical trials is often limited by logistical challenges associated with the handling of biospecimens. Newly developed protocols aim to address these limitations by enabling profiling of fixed tissue. These new solutions need to be benchmarked against well-established protocols to assess their performance and suitability for clinical research. Results We systematically compared different scRNA-seq protocols in a set of samples commonly analysed in clinical trials. The 10X Genomics GEM-X Flex Gene Expression assay (GEM-X Flex), in combination with the “chop-fix” preprocessing protocol, demonstrated superior performance to the standard GEM-X Universal Gene Expression solution when applied to both primary tumor tissue fragments and FFPE blocks. Moreover, the quality of the data obtained from GEM-X Flex applied to FFPE blocks outperformed that of single-nuclei RNA sequencing (snRNA-seq) from frozen biopsies, more robustly capturing the biological signals associated with the mechanism of action of a drug evaluated in an internal clinical trial. Conclusions GEM-X Flex generates reliable, comprehensive transcriptomic data from both fixed tissue and clinical biopsies. By overcoming some of the limitations of fresh and frozen tissue analysis, this protocol offers a robust solution for the broad implementation of scRNA-seq in clinical trials.

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