Nanopore Biopharma Seminar

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 on-target 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. 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.

The success of mRNA vaccines has been realized, in part, due to advances in manufacturing that have enabled the rapid production of billions of doses with sufficient quality and safety. However, mRNA vaccines must be rigorously analyzed to ensure their quality, purity, and integrity, which can otherwise reduce their efficacy and induce side effects. Currently, mRNA vaccines and therapies are analyzed using a range of time-consuming and costly methods. Long-read nanopore sequencing provides a fast, streamlined method to analyze mRNA vaccines and therapies with single-molecule and single-nucleotide resolution. Compared to other industry-standard techniques, nanopore sequencing can comprehensively measure key mRNA quality attributes, including sequence, length, integrity, and purity. We also demonstrate how direct RNA sequencing can analyze mRNA chemistry, including the detection of modified nucleotides. Given these advantages, we anticipate that Oxford nanopore sequencing technologies will be central to the research, development, and manufacture of mRNA vaccines and therapies.

It all started with a hamster—those cute and furry little beasts with cells of gold. Over the course of decades, genetically modified Chinese hamster ovary (CHO) cells have quietly become the workhorse of the industry. They became the boom to biopharmaceutical manufacturing, capable of mass-producing lifesaving therapeutic proteins. At the same time, as molecular characterization methods improved, they likewise became the bane of those tasked to characterize their genome and the magical sequences they have been engineered to hold. Despite the evolution of sequencing technologies, the CHO genome constantly seemed to outmaneuver them, dancing into newer and more complex configurations, all the while creating new and seemingly unending challenges to the task of genome characterization. That is, until now. The advent of longer read sequencing like that offered by Oxford Nanopore Technologies’ platforms, combined with a CRISPR/Cas9 toolbox, have finally provided much-needed relief and a game-changing approach to finally tame the wild CHO genome. Knowing whether your engineered sequence is ‘here’ or ‘there’ in the genome, and why it matters, has never been easier to resolve. Now is the time to learn more about this novel integration site analysis (ISA) approach and how it can fit into your characterization arsenal.