title: Batch effects and the effective design of single-cell gene expression studies.
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.
authors.affiliation: Department of Human Genetics, University of Chicago, Chicago, Illinois, USA.
authors.forename: Po-Yuan
authors.initials: PY
authors.lastname: Tung
authors.affiliation: Department of Human Genetics, University of Chicago, Chicago, Illinois, USA.|Committee on Genetics, Genomics, and Systems Biology, University of Chicago, Chicago, Illinois, USA.
authors.forename: John D
authors.initials: JD
authors.lastname: Blischak
authors.affiliation: Department of Human Genetics, University of Chicago, Chicago, Illinois, USA.
authors.forename: Chiaowen Joyce
authors.initials: CJ
authors.lastname: Hsiao
authors.affiliation: Department of Genetics, Stanford University, Stanford, CA, USA.|Department of Radiology, Stanford University, Stanford, CA, USA.
authors.forename: David A
authors.initials: DA
authors.lastname: Knowles
authors.affiliation: Department of Human Genetics, University of Chicago, Chicago, Illinois, USA.
authors.forename: Jonathan E
authors.initials: JE
authors.lastname: Burnett
authors.affiliation: Department of Genetics, Stanford University, Stanford, CA, USA.|Department of Biology, Stanford University, Stanford, CA, USA.|Howard Hughes Medical Institute, Stanford University, CA, USA.
authors.forename: Jonathan K
authors.initials: JK
authors.lastname: Pritchard
authors.affiliation: Department of Human Genetics, University of Chicago, Chicago, Illinois, USA.|Department of Medicine, University of Chicago, Chicago, Illinois, USA.
authors.forename: Yoav
authors.initials: Y
authors.lastname: Gilad
mesh_terms: D015870:Gene Expression; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D025341:Principal Component Analysis; D012313:RNA / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017423:Sequence Analysis, RNA; D059010:Single-Cell Analysis
chemical_list: D012313:RNA