Identification of high-confidence human poly(A) RNA isoform scaffolds using nanopore sequencing

Area of Science:

• Molecular Biology
• Genomics
• Transcriptomics

Background:

• Nanopore sequencing offers direct RNA analysis, enabling exon linkage and modification detection.
• Conventional methods struggle to unambiguously identify 5' and 3' ends of polyadenylated (poly(A)) RNA.
• RNA degradation complicates the precise identification of transcription start and end sites.

Purpose of the Study:

• To identify individual full-length human RNA isoforms using direct RNA nanopore sequencing.
• To overcome limitations in determining RNA 5' and 3' ends in nanopore sequencing data.
• To discover novel RNA isoforms and improve transcriptomic analysis.

Main Methods:

• Developed an oligomer adaptation method to replace the biological cap with a modified one for improved 5' end sequencing.
• Screened 5'-capped nanopore reads for features indicative of 3' polyadenylation sites.
• Combined cap identification and polyadenylation site screening to define full-length RNA scaffolds.

Main Results:

• Identified 294,107 high-confidence full-length RNA scaffolds from human cells.
• The majority of scaffolds (257,721) aligned to known protein-coding genes.
• Discovered 4,876 unannotated RNA isoforms, often internal to longer known transcripts.
• Orthogonal data (CAGE, DNase-HS seq) validated the identified RNA scaffolds.

Conclusions:

• The oligomer adaptation method enables unambiguous identification of 5' capped ends in direct RNA nanopore sequencing.
• This approach successfully identifies full-length RNA isoforms, including novel ones.
• The findings significantly advance the ability to comprehensively analyze the human transcriptome.