Article series: Single-cell omics

Computational and analytical challenges in single-cell transcriptomics

Journal name:
Nature Reviews Genetics
Year published:
Published online


The development of high-throughput RNA sequencing (RNA-seq) at the single-cell level has already led to profound new discoveries in biology, ranging from the identification of novel cell types to the study of global patterns of stochastic gene expression. Alongside the technological breakthroughs that have facilitated the large-scale generation of single-cell transcriptomic data, it is important to consider the specific computational and analytical challenges that still have to be overcome. Although some tools for analysing RNA-seq data from bulk cell populations can be readily applied to single-cell RNA-seq data, many new computational strategies are required to fully exploit this data type and to enable a comprehensive yet detailed study of gene expression at the single-cell level.

At a glance


  1. Comparison of bulk and scRNA-seq analytical strategies.
    Figure 1: Comparison of bulk and scRNA-seq analytical strategies.

    A flow chart of the steps in analysis of high-throughput RNA sequencing (RNA-seq) data from bulk cell populations and from single cells is shown. Methods that are common to both approaches are shown in purple, whereas key differences in analysis methods between bulk-based RNA-seq and single-cell RNA-seq (scRNA-seq) are shown in blue and red, respectively. FPKM, fragments per kilobase of exon per million fragments mapped; PCA, principal component analysis.

  2. Quality control and normalization.
    Figure 2: Quality control and normalization.

    a | Basic quality control steps are shown. After generating single-cell RNA sequencing (scRNA-seq) data, a key first step is to assess the quality of the data. In addition to quality metrics developed for bulk RNA-seq, it is important to determine whether cells have been captured efficiently and the mRNA fraction amplified faithfully. Two simple but important criteria are to compare the percentage of unmapped reads and the percentage of reads mapped to the external spike-in molecules across cells. Cells in which either of these values is high (grey) are of poor quality and should be discarded, leaving only the higher-quality cells (green) for downstream analyses. b | Spike-ins can be used to model technical variability and examine relative variability in cell size for non-unique molecular identifier (UMI)-based scRNA-seq data. If external spike-in molecules are added at the same volume to the RNA mixture from each cell before processing, they can be used to quantify the degree of technical variability across cells and to examine the relationship between technical variation and gene expression (upper panel). The x axis shows average expression levels across cells, and the y axis shows the squared coefficient of variation; blue points are extrinsic spike-in molecules. The red line indicates the fitted relationship between technical noise and gene expression strength. Additionally, by calculating the ratio between the numbers of reads mapped to the spike-in sequences and to the genes from the organism of interest, the relative amount of mRNA contained in each cell can be estimated (lower panel). c | Spike-ins can also be used to model technical variability and to examine relative variability in cell size for UMI-based scRNA-seq data. Similar to part b, the upper panel illustrates the relationship between technical noise and expression strength — the difference is that the expression level of each gene is now quantified as the number of unique cDNA molecules. Additionally, spike-ins can be used to quantify the capture efficiency and thus infer the number of mRNA molecules contained in the lysate of each cell (lower panel). Upper panels of parts b and c adapted from Ref. 33 and Ref. 40, respectively, Nature Publishing Group.

  3. Confounding variables and how to account for them.
    Figure 3: Confounding variables and how to account for them.

    a | For each gene, the observed expression profile generated from single-cell RNA sequencing (scRNA-seq) is caused by a combination of factors. For example, if cells are being sampled randomly from a mixed population containing naive (that is, undifferentiated) cells and cells that are closer to being fully differentiated, then for each cell, the expression profile is a combination of a variety of factors (including position on the differentiation cascade, cell cycle state and apoptotic state). Factors such as the cell cycle or apoptotic state can be considered confounders that prevent the signal of interest (the differentiation state of a cell) from being uncovered. b | Confounding factors need to be identified and corrected for in downstream analyses. Latent-variable models, which are built on approaches applied in bulk RNA-seq studies to infer and correct for hidden factors that cause gene expression heterogeneity56, 57, 59, can be used to deduce the correlation between cells due to factors such as the cell cycle or apoptotic state. Subsequently, the extent of variance in the expression of each gene across cells that is attributable to this factor (and other factors) can be inferred. Additionally, the scRNA-seq data can be corrected by using regression analyses to remove the confounding factor, thus facilitating downstream analyses such as clustering or network analyses. Figure from Ref. 61, Nature Publishing Group.

  4. Finding new cell types and allocating cells along a differentiation cascade.
    Figure 4: Finding new cell types and allocating cells along a differentiation cascade.

    Unbiased clustering approaches based on principal component analysis (PCA)-like methods62, 63, 66 can be used on a mixed population of cells, to either map them along a differentiation cascade or cluster them into new cell types55, 58. Subsequently, the newly identified cascades or populations can be characterized, and new marker genes can be found by identifying genes or transcript isoforms that are differentially expressed between the populations.

  5. The kinetics of transcription.
    Figure 5: The kinetics of transcription.

    a | Single-cell RNA sequencing (scRNA-seq) can be used to study the kinetics of transcription. RNA labelling followed by pulse microscopy (left panel) can be used to track the expression of a gene over time82. scRNA-seq can be used to obtain an instantaneous snapshot of this distribution by measuring the expression of an individual gene across many cells (middle panel)81. Subsequently, these data can be used to draw inferences about the kinetics of transcription. b | Allele-specific expression can be studied using scRNA-seq. Allele-specific expression can be assayed using single-nucleotide polymorphisms (SNPs) in the sequence of a transcript to allocate reads to alternative alleles. Subsequently, the number of cells in which both alleles are expressed and the numbers of cells in which allele 1 or allele 2 is exclusively expressed can be counted. This allows the identification of genes that display evidence of monoallelic expression4. One important challenge is to address technical issues, especially allelic dropout during sample preparation, which can bias the results.


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Author information


  1. European Molecular Biology Laboratory European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SD, UK.

    • Oliver Stegle,
    • Sarah A. Teichmann &
    • John C. Marioni
  2. Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.

    • Sarah A. Teichmann &
    • John C. Marioni

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The authors declare no competing interests.

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Author details

  • Oliver Stegle

    Oliver Stegle is a group leader at the European Molecular Biology Laboratory European Bioinformatics Institute (EMBL-EBI) in Cambridge, UK. His group develops statistical methods to analyse high-dimensional molecular traits in different contexts. He received his Ph.D. from the University of Cambridge, UK, in physics in 2009, working with David MacKay. After a period as a postdoctoral researcher at the Max Planck Campus in Tübingen, Germany, he moved to the EMBL-EBI in 2012 to establish his own research group.

  • Sarah A. Teichmann

    Sarah A. Teichmann is a group leader at the European Molecular Biology Laboratory European Bioinformatics Institute (EMBL-EBI) and Wellcome Trust Sanger Institute in Cambridge, UK. Her group studies global gene regulation with a focus on CD4+ T cells, and the assembly and evolution of protein complexes. She received her Ph.D. in computational genomics from the Medical Research Council (MRC) Laboratory of Molecular Biology, Cambridge, UK, where she worked with Cyrus Chothia, and was a Beit Memorial Fellow at University College London, UK, with Janet Thornton. From 2001 to 2012 she was an MRC programme leader at the MRC Laboratory of Molecular Biology. She moved to the EMBL-EBI and Wellcome Trust Sanger Institute in 2013.

  • John C. Marioni

    John C. Marioni is a group leader at the European Molecular Biology Laboratory European Bioinformatics Institute (EMBL-EBI) and an associate faculty member at the Wellcome Trust Sanger Institute in Cambridge, UK. His group develops computational methods for understanding the regulation of gene expression in the context of evolution and development, with a particular focus on investigating variability in expression (and other molecular traits) between individual cells. He received his Ph.D. from the University of Cambridge, UK, in applied mathematics, working with Simon Tavaré. After a period of postdoctoral research at the University of Chicago, Illinois, USA, under the supervision of Matthew Stephens, he moved to the EMBL-EBI in 2010 to establish his own research group. John C. Marioni's homepage.

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