Aziz Alafage from Broad Clinical Labs presents two ambitious applications leveraging Rosh's new SVX platform for advanced RNA research. The first project involves high-scale multiomic drug response profiling in multiplex PRISM cancer cell lines. This study combines single-cell RNA sequencing (using 10x Flex) with over 320 protein expression targets, all sequenced on the SPX platform to understand KRAS inhibitor resistance. The resulting "phenomenal" data allowed detailed classification of drug responses, survival outcomes, and associated mutation enrichments, consistent with existing literature. The second project explores ultra-high throughput single-cell RNA isoform sequencing, demonstrating the SPX's capability to deliver read lengths (predominantly 500-800 base pairs) sufficient for spanning multiple splice junctions and resolving full-length isoforms, leading to the exciting identification of novel, unannotated isoforms. This work underscores the transformative potential of the SVX/SPX platform for fundamental biological discovery and drug development.
Exploring Multiomic Drug Response and RNA Isoform Sequencing with Rosh's SVX/SPX Platform
0:00 Introduction: Aziz Alafage (Director of Molecular R&D at Broad Clinical Labs) introduces efforts to deploy Rosh's new SVX platform for ambitious RNA research.
0:43 Main Research Areas: Two primary areas of focus are outlined:
High-scale multiomic drug response profiling in multiplex PRISM cell lines.
Ultra-high throughput single-cell RNA isoform sequencing using the SPX platform.
1:08 KRAS Drug Resistance: The presentation details the challenge of therapeutic evasion and drug resistance in treating KRAS-mutated cancers, a target of small molecule inhibitors. The goal is to understand these evasion mechanisms to develop combination therapies.
1:51 PRISM Partnership: A collaboration with Broad's PRISM team utilizes multiplex cancer cell lines, consisting of 500 unique co-cultured lines identifiable via their expressed PRISM barcodes, enabling efficient profiling of drug effects across diverse cancers.
2:16 Experimental Design: PRISM pools were treated with RAS inhibitors (BA 2865, RMC 6236) and controls. Single-cell analysis was performed at 3 and 6 hours, complemented by a 5-day survival assay to link outcomes to early drug responses.
3:14 Multiomic Readouts: The experiment involved multiomic single-cell profiling, capturing PRISM barcodes for cell line identification, protein expression (over 320 targets using the Protein Tex Discovery panel), and whole transcriptome data via the 10x Flex single-cell platform, all sequenced on the SPX platform (yielding 80 billion reads).
4:03 High-Quality Data: The generated data was described as "phenomenal," with a median of 1000 cells per cell line, 28,000 unique molecular identifiers (UMIs) and 7,000 genes per cell, exhibiting high quality with no batch effects and strong correlation with orthogonally derived bulk RNA-seq data.
5:35 Impressive Protein Data: Protein expression data was highly effective, allowing resolution of many cell lines based on protein expression alone, with approximately 13,000 protein UMIs and a median of 300 proteins detected per cell.
5:58 Utility of RNA & Protein Capture: The presentation emphasizes the value of capturing both RNA and protein expression, as their correlation varies (e.g., transcription factors are highly post-transcriptionally regulated), highlighting the necessity of multiomic approaches for comprehensive biological understanding.
7:09 Diverse Drug Responses: A range of transcriptional responses to drug inhibitors was observed, visualized through colocalization analysis, illustrating varied cellular reactions.
7:57 Survival Outcomes & Mutation Analysis: Cell lines were categorized into four survival classes (sensitive to both drugs, sensitive to RMC only, sensitive to BI only, resistant to both). Unique mutation enrichments, such as KRAS in sensitive cells and BRAF in resistant cells, were identified and found to be concordant with scientific literature.
9:24 Future Outlook for Drug Response: The initial analysis revealed vast potential for discovery. Future work will integrate classical analyses with advanced AI approaches to uncover complex biological signals, aiming for a high-yield strategy to systematically identify and validate resistance mechanisms for drug development.
10:05 RNA Isoform Sequencing with SPX: The second project focuses on ultra-high throughput RNA isoform sequencing using the SPX platform.
10:40 SPX Read Length Capabilities: The SPX platform delivered read lengths predominantly between 500 and 800 base pairs, with a significant long tail. Analysis confirmed these lengths are sufficient to span multiple splice junctions and resolve full-length isoforms, showing a sharp increase in "full splice match" reads beyond 300 base pairs.
11:52 Discovery of Novel Isoforms: The platform successfully identified an unannotated isoform of the CD1C gene, demonstrating its potential for novel biological discoveries.
12:17 Acknowledgments: The speaker acknowledges the immense talent of his group, the PRISM team, Broad Clinical Labs, and industry partners, praising the collaborative effort and recognizing Rosh's technology as transformative.