RACE-Nano-Seq: Profiling Transcriptome Diversity of a Genomic Locus.

Area of Science:

• Molecular Biology
• Genomics
• Transcriptomics

Background:

• The human transcriptome is complex, challenging complete annotation.
• Traditional RNA-sequencing (RNA-seq) methods struggle with low-abundance transcripts and isoform resolution due to sensitivity and read length limitations.
• Long-read sequencing offers full-length transcript information but faces throughput constraints for capturing rare transcripts.

Purpose of the Study:

• To introduce RACE-Nano-Seq, a targeted RNA enrichment strategy coupled with Nanopore sequencing.
• To enable comprehensive and accurate profiling of low-abundance and complex transcript isoforms.
• To facilitate the discovery of novel transcripts and alternative splicing events.

Main Methods:

• RACE-Nano-Seq utilizes inverse PCR with primers targeting specific anchor sequences (regions of interest).
• This approach enriches for corresponding transcripts in both 5' and 3' directions.
• The method is scalable for high-throughput transcriptome profiling via multiplexing.

Main Results:

• RACE-Nano-Seq accurately profiles low-abundance transcripts, revealing complex transcript profiles at targeted loci.
• The protocol demonstrates high sensitivity for detecting transcripts.
• It enables the characterization of complex genomic loci, including the discovery of novel transcripts and exons.

Conclusions:

• RACE-Nano-Seq overcomes limitations of traditional RNA-seq and long-read sequencing for transcriptomic analysis.
• This method provides a sensitive and comprehensive approach for studying transcriptome complexity.
• The protocol is adaptable for various research scales and can be performed in standard molecular biology labs.