FIRST-seq: a nanopore-based cDNA sequencing platform for RNA modification and structure profiling

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• RNA modifications are crucial for cellular function and gene regulation.
• Existing methods for RNA modification detection have limitations in resolution, cost, or enzyme compatibility.
• Reverse transcription (RT) errors can be leveraged to map RNA modifications.

Purpose of the Study:

• To develop a flexible, cost-effective nanopore cDNA sequencing method for RNA modification detection.
• To enable single-nucleotide resolution analysis of RT signatures for various RNA modifications.
• To optimize conditions for accurate RNA modification profiling using long-read sequencing.

Main Methods:

• Developed FIRST-seq, a nanopore cDNA method avoiding second-strand synthesis and PCR.
• Benchmarked multiple reverse transcription (RT) enzymes and buffers to optimize error detection and minimize premature termination.
• Integrated FIRST-seq with Dimethyl sulfate (DMS) probing for detecting specific RNA modifications.

Main Results:

• Identified optimal RT enzymes and buffers for enhanced error detection and reduced premature termination.
• Demonstrated FIRST-seq's ability to accurately detect m1A and m3C modifications at unpaired sites.
• Successfully recapitulated known RNA structures in vitro and in vivo using FIRST-seq coupled with DMS probing.

Conclusions:

• FIRST-seq is a versatile and cost-effective platform for profiling RNA modifications using long-read sequencing.
• The method enables accurate detection of chemical-induced and natural RNA modifications.
• FIRST-seq facilitates the study of RNA structure and function through precise modification mapping.