Suppression of artifacts and barcode bias in high-throughput transcriptome analyses utilizing template switching

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Template switching (TS) is a reverse transcriptase mechanism used in transcriptome analysis.
• TS offers a simple protocol, minimizing sample loss, ideal for limited RNA amounts like single cells.
• TS enables DNA barcode/index introduction for multiplexing and increased sequencing throughput.

Purpose of the Study:

• To identify and characterize artifacts arising from template switching.
• To address biases introduced by strand invasion during TS-based barcoding.
• To develop computational and experimental solutions for TS-related artifacts.

Main Methods:

• Investigated template switching artifacts, specifically those related to strand invasion.
• Developed an in silico strategy to eliminate identified artifacts.
• Proposed an experimental method to suppress biases from template switching.

Main Results:

• Discovered that strand invasion during TS-based barcoding introduces unsystematic biases.
• Successfully eliminated TS artifacts using an in silico strategy.
• Developed an experimental solution to mitigate TS-induced biases.

Conclusions:

• Strand invasion is a significant source of bias in TS-based transcriptome analysis.
• In silico and experimental strategies can effectively correct and suppress TS artifacts.
• These solutions enhance the accuracy and reliability of RNA sequencing methods employing template switching.