(642g) Rapid Diagnosis of Plasmid Transcriptions with Nanopore Sequencing

Transcriptomics analysis provides valuable insights into cellular processes by revealing the dynamic profile of genes involved in product formation and stress responses. High-throughput sequencing, such as Illumina-based workflows, has been the primary method for the past decade. However, long turnaround times from service providers and the necessity of ribosomal RNA depletion have significantly increased the cost of performing transcriptomics analysis within each design-build-test cycle. This situation motivated us to adopt nanopore sequencing as it has become an attractive alternative for small laboratories to conduct sequencing work. In this study, we have developed a direct cDNA nanopore sequencing workflow with an mRNA enrichment strategy. Oligonucleotides are used to block the polyadenylation of rRNA. Up to 6 samples can be pooled to analyze plasmid-based transcriptions on a Flongle Flow Cell without compromising the resolution in data analysis. The same setup can also be used to quantify the top transcribed genes in the global transcriptome, although the number of multiplexed samples needs to be reduced. The entire workflow can be completed within 2-3 days, making RNA sequencing accessible in terms of both time and cost. As a proof of concept, RNA sequencing is being used to optimize carotenoids production in Escherichia coli. We observed highly varied transcription profiles within the genes, along with transcriptional defects such as intragenic terminators. These results were cross-validated using qualitative PCR. The insights gained from RNA sequencing can guide us in the rational redesign of the involved plasmids. Our described sequencing workflow can be implemented to diagnose issues in day-to-day strain engineering work.