Abstract
Purpose :
Alternative RNA splicing (AS) plays an important role in the process of retinal development. Although short-read next generation RNA sequencing can provide some insights into AS, long-read sequencing (LRS) technologies, which sequence full-length mRNA molecules, allow for more comprehensive splicing analysis. The goal of this study is to use nanopore-based LRS to measure isoform expression and characterize AS at the single cell level in differentiating human stem cell-derived retinal organoids (ROs).
Methods :
Human induced pluripotent stem cells were differentiated into 3D ROs using an established protocol. ROs were dissociated at time points during differentiation for single cell mRNA capture using droplet-based platforms. An optimized version of the rolling circle to concatemeric consensus (R2C2) method was used to improve read accuracy of full-length, single cell cDNA. R2C2 cDNA was sequenced using the Oxford Nanopore Technologies (ONT) ligation sequencing kit and PromethION flow cells. Cell barcodes were demultiplexed and cell specific reads were assigned using C3POa. Cell clustering was performed with Seurat, and transcript quantification of the full-length reads from each cell were generated with Salmon.
Results :
The C3POa pipeline demultiplexed over 90% of total ONT reads. UMAP clustering of R2C2 ONT data resulted in distinct clusters representing the expected RO cell types, including retinal neurons, glia, and both cycling and non-cycling progenitor cells. Multiple gene isoforms were observed across these cell types. For example, three of eight protein-coding transcripts (ENSEMBL annotated) of PRDM1 (also known as BLIMP1) were detected exclusively in retinal neurons. Of these, the PRDM1β variant, which utilizes an alternative promoter, was predominantly expressed in the neurons. Several Basigin (BSG) transcripts were also detected, including a retinal-specific isoform that encodes for the receptor for the rod-derived cone viability factor, as well as both protein-coding and retained intron transcripts in glial and progenitor cells. Other highly spliced genes, like Neurofibromin (NF1), showed a large diversity of expressed transcripts in the ROs in a cell-type specific manner.
Conclusions :
R2C2 ONT sequencing reads can successfully generate isoform-level transcriptomes of ROs at the single cell level. Studies comparing isoform expression in ROs across multiple differentiation time points are ongoing.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.