Rachel Thijssen from the Walter and Eliza Hall Institute of Medical Research and Australia & Amsterdam University Medical Centers in the Netherlands presented at London Calling 2023 on “Single-cell long-read RNA sequencing reveals complex heterogeneity.” Thijssen spoke about the hallmarks of cancer and cells not responding to signals appropriately. BCL2 can be targeted in chronic lymphocytic leukemia (CLL). Most patients respond to Venetoclax (VEN), but after a period of time, cancer cells become resistant in some cases. After bulk sequencing, a BCL2 mutation in CLL was identified. Single-cell transcriptomics with the 10X Genomics system is powerful, but limited by short reads. Thijssen and colleagues developed a multi omics approach that uses subsampling for whole transcriptome. A second approach they developed is to use cDNA for rapid capture hybridization sequencing RaCH-seq. For this, a probe panel is designed: 20 bp probes for exons. The probes are biotinylated and hybridized with cDNA for capture and amplification. An engineered overhang and an analysis pipeline called Flames were developed. Flames pipeline performs quality control and identifies the barcodes before aligning. To investigate Venetoclax resistance, single-cell RaCH-seq and a panel for enrichment and sequencing was developed. Tumor single cell data indicated that cells with the mutation cluster together. Furthermore, a cluster of cells express a new pro-apoptotic NOXA transcript. Then, by integrating short- and long-read sequencing, the team identified “at least 4 distinct mechanisms driving VEN resistance.” Using already stored cDNA libraries, single-cell transcriptomics using Nanopore sequencing allowed for identification of mutations in single-cells. The single-cell transcriptomics methods and combination of short- and long-read sequencing are complex and powerful. The ability to go back to cDNA from stored samples and investigate a panel of genes/sequences with long-read can be very useful!
