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Normalization and variance stabilization of single-cell RNA-seq data using regularized negative binomial regression

View ORCID ProfileChristoph Hafemeister, View ORCID ProfileRahul Satija
doi: https://doi.org/10.1101/576827
Christoph Hafemeister
1New York Genome Center, New York City, NY, USA
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Rahul Satija
1New York Genome Center, New York City, NY, USA
2Center for Genomics and Systems Bioligy, New York University, New York City, NY, USA
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Abstract

Single-cell RNA-seq (scRNA-seq) data exhibits significant cell-to-cell variation due to technical factors, including the number of molecules detected in each cell, which can confound biological heterogeneity with technical effects. To address this, we present a modeling framework for the normalization and variance stabilization of molecular count data from scRNA-seq experiments. We propose that the Pearson residuals from ’regularized negative binomial regression’, where cellular sequencing depth is utilized as a covariate in a generalized linear model, successfully remove the influence of technical characteristics from downstream analyses while preserving biological heterogeneity. Importantly, we show that an unconstrained negative binomial model may overfit scRNA-seq data, and overcome this by pooling information across genes with similar abundances to obtain stable parameter estimates. Our procedure omits the need for heuristic steps including pseudocount addition or log-transformation, and improves common downstream analytical tasks such as variable gene selection, dimensional reduction, and differential expression. Our approach can be applied to any UMI-based scRNA-seq dataset and is freely available as part of the R package sctransform, with a direct interface to our single-cell toolkit Seurat.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted March 18, 2019.
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Normalization and variance stabilization of single-cell RNA-seq data using regularized negative binomial regression
Christoph Hafemeister, Rahul Satija
bioRxiv 576827; doi: https://doi.org/10.1101/576827
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Normalization and variance stabilization of single-cell RNA-seq data using regularized negative binomial regression
Christoph Hafemeister, Rahul Satija
bioRxiv 576827; doi: https://doi.org/10.1101/576827

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