Sereti, K. | bioRxiv Science

bioRxiv2023

Accurate long-read transcript discovery and quantification at single-cell resolution with Isosceles

Accurate detection and quantification of mRNA isoforms from nanopore long-read sequencing remains challenged by technical noise, particularly in single cells. To address this, we introduce Isosceles, a computational toolkit that outperforms other methods in isoform detection sensitivity and quantification accuracy across single-cell, pseudo-bulk and bulk resolution levels, as demonstrated using synthetic and biologically-derived datasets. Isosceles improves the fidelity of single-cell transcriptome quantification at the isoform-level, and enables flexible downstream analysis. As a case study, we apply Isosceles, uncovering coordinated splicing within and between neuronal differentiation lineages. Isosceles is suitable to be applied in diverse biological systems, facilitating studies of cellular heterogeneity across biomedical research applications.

bioinformatics↗

bioRxiv2023

Single-cell long-read targeted sequencing reveals transcriptional variation in ovarian cancer

Single-cell RNA sequencing predominantly employs short-read sequencing to characterize cell types, states and dynamics; however, it is inadequate for comprehensive characterization of RNA isoforms. Long-read sequencing technologies enable single-cell RNA isoform detection but are hampered by lower throughput and unintended sequencing of artifacts. Here we developed Single-cell Targeted Isoform Long-Read Sequencing (scTaILoR-seq), a hybridization capture method which targets over a thousand genes of interest, improving the median number of unique transcripts per cell by 29-fold. We used scTaILoR-seq to identify and quantify RNA isoforms from ovarian cancer cell lines and primary tumors, yielding 10,796 single-cell transcriptomes. Using long-read variant calling we revealed associations of expressed single nucleotide variants (SNVs) with alternative transcript structures. In addition, phasing of SNVs across transcripts facilitated measurement of allelic imbalance within distinct cell populations. Overall, scTaILoR-seq is a long-read targeted RNA sequencing method and analytical framework for exploring transcriptional variation at single-cell resolution.

genomics↗