Exploring temperature-dependent transcriptomic adaptations in Yersinia pestis using direct cDNA sequencing by Oxford...

Area of Science:

• Microbiology
• Genomics
• Molecular Biology

Background:

• Transcriptomics is crucial for understanding bacterial pathogen adaptation and disease mechanisms.
• Current transcriptomic methods face challenges including cost, technical complexity, and biosafety concerns, particularly for virulent pathogens.

Purpose of the Study:

• To develop a streamlined, cost-effective, and in-house implementable RNA-Sequencing (RNA-Seq) workflow for bacterial transcriptomics.
• To apply this workflow to Yersinia pestis to enhance operon discovery, genome annotation, and gene regulation analysis.

Main Methods:

• Utilized Oxford Nanopore Technologies for direct cDNA sequencing, bypassing PCR amplification to reduce bias.
• Implemented multiplexing capabilities and integrated quality control and alignment benchmarking.
• Applied the workflow to Yersinia pestis, performing transcriptomic profiling at 21°C and 37°C.

Main Results:

• Generated an experimentally validated operon map for Yersinia pestis, identifying novel transcriptional units.
• Revealed temperature-driven metabolic shifts, including the upregulation of sulfur metabolism and the dmsABCD operon.
• Demonstrated the workflow's suitability for high-risk or understudied bacterial pathogens.

Conclusions:

• The developed long-read RNA-Seq workflow offers a practical solution for bacterial transcriptomics, overcoming limitations of existing methods.
• This approach facilitates operon discovery, genome annotation, and the study of gene regulation in challenging pathogens.
• Provides valuable insights into Yersinia pestis adaptation mechanisms relevant to plague pathogenesis.