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

Christoph Hafemeister, Rahul Satija

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish (US)
Article number296
JournalGenome biology
Issue number1
StatePublished - Dec 23 2019


  • Normalization
  • Single-cell RNA-seq

ASJC Scopus subject areas

  • Ecology, Evolution, Behavior and Systematics
  • Genetics
  • Cell Biology


Dive into the research topics of 'Normalization and variance stabilization of single-cell RNA-seq data using regularized negative binomial regression'. Together they form a unique fingerprint.

Cite this