Batch effects and the effective design of single-cell gene expression studies

Sci Rep. 2017 Jan 3;7:39921. doi: 10.1038/srep39921.

Abstract

Single-cell RNA sequencing (scRNA-seq) can be used to characterize variation in gene expression levels at high resolution. However, the sources of experimental noise in scRNA-seq are not yet well understood. We investigated the technical variation associated with sample processing using the single-cell Fluidigm C1 platform. To do so, we processed three C1 replicates from three human induced pluripotent stem cell (iPSC) lines. We added unique molecular identifiers (UMIs) to all samples, to account for amplification bias. We found that the major source of variation in the gene expression data was driven by genotype, but we also observed substantial variation between the technical replicates. We observed that the conversion of reads to molecules using the UMIs was impacted by both biological and technical variation, indicating that UMI counts are not an unbiased estimator of gene expression levels. Based on our results, we suggest a framework for effective scRNA-seq studies.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Gene Expression
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Induced Pluripotent Stem Cells / cytology
  • Induced Pluripotent Stem Cells / metabolism
  • Principal Component Analysis
  • RNA / chemistry
  • RNA / isolation & purification
  • RNA / metabolism*
  • Sequence Analysis, RNA
  • Single-Cell Analysis*

Substances

  • RNA