Multicellular, IVT-derived, unmodified human transcriptome for nanopore-direct RNA analysis.

Area of Science:

• Molecular Biology
• Genomics
• Bioinformatics

Background:

• Nanopore direct RNA sequencing (DRS) allows for the detection of RNA modifications.
• Modification-free transcripts are essential controls for accurate DRS analysis, providing a baseline measurement.
• Generating these controls can be complex and may introduce nanopore-specific biases.

Purpose of the Study:

• To produce and characterize modification-free RNA transcripts for use as controls in nanopore DRS.
• To establish a baseline for canonical nucleotide measurements within a biologically relevant context.
• To provide a community resource for RNA modification analysis in human cell lines.

Main Methods:

• Produced direct RNA sequencing datasets using modification-free transcripts.
• Transcripts were generated via in vitro transcription (IVT) of cDNA from six human cell lines.
• Characterized variations across different cell lines to understand their impact on analysis.

Main Results:

• Generated high-quality nanopore DRS datasets from modification-free human transcripts.
• Identified and characterized cell-line-specific variations in the control transcripts.
• Demonstrated the utility of these datasets for interpreting RNA modification analyses.

Conclusions:

• Modification-free transcripts from in vitro transcription serve as valuable controls for nanopore DRS.
• Understanding cell-line variation is crucial for accurate interpretation of RNA modification data.
• These datasets offer a standardized resource to enhance the reliability of RNA modification studies.