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Molecular spikes: a gold standard for single-cell RNA counting
Christoph Ziegenhain1, Gert-Jan Hendriks1, Michael Hagemann-Jensen1
1Department of Cell and Molecular Biology, Karolinska Institute, Stockholm, Sweden.
Nature Methods
|April 26, 2022
Summary
New molecular spikes enable accurate RNA counting in single-cell RNA sequencing (scRNA-seq). This method identifies and corrects issues with unique molecular identifier (UMI) counts, improving data reliability.
Area of Science:
- Molecular Biology
- Genomics
- Bioinformatics
Background:
- Single-cell RNA sequencing (scRNA-seq) relies on accurate molecule counting to address amplification biases.
- Existing methods lack an experimental strategy to evaluate RNA counting performance.
Purpose of the Study:
- Introduce molecular spikes for evaluating RNA counting accuracy in scRNA-seq.
- Identify critical experimental and computational conditions affecting RNA counting.
- Develop strategies to correct impaired RNA counting experiments.
Main Methods:
- Developed RNA spike-ins with built-in unique molecular identifiers (UMIs).
- Utilized molecular spikes to assess RNA counting performance across different scRNA-seq conditions.
- Applied the UMIcountR R package for data analysis and correction.
Main Results:
- Discovered impaired RNA counting in certain scRNA-seq methods due to inflated UMI counts.
- Demonstrated that molecular spikes can identify conditions with inaccurate RNA abundance estimates.
- Successfully improved estimates of total endogenous RNA amounts using molecular spikes.
Conclusions:
- Molecular spikes provide a crucial tool for validating new scRNA-seq methods.
- This approach enables better estimation and correction of cellular mRNA amounts.
- Facilitates a more in-depth characterization of RNA counting in scRNA-seq experiments.
