Comprehensive multi-center assessment of small RNA-seq methods for quantitative miRNA profiling

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• RNA sequencing is a key tool for small RNA profiling in various biological samples.
• Existing small RNA library preparation methods lack systematic validation for accuracy and reproducibility.
• Understanding biases in small RNA-seq is crucial for reliable quantitative analysis.

Purpose of the Study:

• To systematically evaluate the accuracy and reproducibility of different small RNA sequencing library preparation methods.
• To identify and characterize protocol- and sequence-specific biases in small RNA-seq.
• To assess the impact of library preparation on microRNA quantification and adenosine-to-inosine editing detection.

Main Methods:

• A consortium of nine laboratories independently sequenced reference samples of synthetic small RNAs and human plasma-derived RNA.
• Three commercial library preparation methods using defined adapters and six using degenerate base adapters were assessed.
• Performance was evaluated based on accuracy, reproducibility, and identification of biases, including those affecting microRNA editing.

Main Results:

• Both protocol- and sequence-specific biases were identified across tested small RNA-seq library preparation methods.
• Biases were found to reduce the accuracy of detecting adenosine-to-inosine editing in microRNAs.
• Library preparation methods incorporating degenerate base adapters demonstrated mitigation of identified biases.
• Relative microRNA quantification between samples using small RNA-seq proved accurate and reproducible across laboratories and methods.

Conclusions:

• Small RNA sequencing library preparation methods exhibit significant biases that can affect quantitative profiling.
• The use of degenerate base adapters in library preparation is recommended to improve accuracy and reproducibility.
• Despite identified biases, microRNA relative quantification via small RNA-seq is robust and reproducible across diverse methods and laboratories.