RT Journal Article
SR Electronic
T1 Variable RNA sampling biases mediate concordance of single-cell and nucleus sequencing across cell types
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2022.08.01.502392
DO 10.1101/2022.08.01.502392
A1 John T. Chamberlin
A1 Younghee Lee
A1 Gabor T. Marth
A1 Aaron R. Quinlan
YR 2022
UL http://biorxiv.org/content/early/2022/08/03/2022.08.01.502392.abstract
AB The capacity for nuclear RNA measurements to recapitulate results from whole cells is essential to the utility of single-nucleus RNA-seq. While early studies argued that nuclear samples could yield comparable results to single-cell RNA-seq, both assays have since been appreciated to be subject to certain systematic biases. These include differences in the localization and capture mode of spliced and unspliced RNAs. However, variability in these biases across cell types has received limited direct attention. Here, we describe the contrasting effects of mRNA and pre-mRNA sampling on inter-assay compatibility among cell types of the cortex, their mechanisms, and their impact on the generalizability of a recently published normalization method. We show that baseline similarity is comparatively high in cortex cell types, and is moderated both by gene length bias in pre-mRNAs and by inherent differences in mRNA abundances, the balance of which varies considerably across cell types. These factors cause inconsistent performance of the normalization method, which emphasizes mRNA measurements by downweighting pre-mRNA measurements according to gene length. Critically, the high baseline similarity of single-cell and nucleus RNA-seq in cortex depends on the inclusion of pre-mRNA signal, but pre-mRNA abundances alone are not strongly predictive of mRNA abundances. This is important to problems such as bulk sample deconvolution. Broadly, our analysis provides a mechanistic explanation for variation in assay similarity across cell types and argues for the potential application of post hoc normalization approaches as an alternative route to improved biological interpretation.Competing Interest StatementThe authors have declared no competing interest.