Low-bias, strand-specific transcriptome Illumina sequencing by on-flowcell reverse transcription (FRT-seq)

Area of Science:

• Molecular Biology
• Genomics
• Next-Generation Sequencing

Background:

• Second-generation sequencing relies on clonal amplification of single template strands on a solid surface.
• Library preparation typically involves PCR, an amplification method known for introducing biases that reduce data acquisition efficiency.

Purpose of the Study:

• To develop a transcriptome sequencing method that eliminates PCR-induced biases and duplicates.
• To enhance the efficiency, reproducibility, and data quality of transcriptome sequencing on Illumina platforms.

Main Methods:

• Developed flowcell reverse transcription sequencing, performing reverse transcription directly on the flowcell using unamplified, adapter-ligated mRNA.
• Applied this method to transcriptome sequencing for Illumina sequencers.

Main Results:

• Successfully removed PCR biases and duplicates from sequencing data.
• Generated highly reproducible, strand-specific paired-end data.
• Demonstrated a rapid procedure, generating clusters from mRNA in 2 days.

Conclusions:

• Flowcell reverse transcription sequencing offers a more accurate and reproducible alternative to traditional PCR-based library preparation.
• This method significantly improves transcriptome data quality by mitigating amplification biases.
• The streamlined protocol accelerates the sequencing workflow, enabling faster data generation.