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Towards progress in batch effects and biases in single-cell RNA-Seq data Stephanie Hicks Dana-Farber Cancer Institute / Harvard SPH @stephaniehicks Single Cell Genomics 2016 Wellcome Genome Campus

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Bimodal distribu=on of expression Number of Cells Expression level of Gene X μ 1 μ 2 True Expression

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Bimodal distribu=on of expression Expression level of Gene X μ 1 μ 2 True Expression Observed Expression Number of Cells

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Patel et al. (2014) Science Cells cluster by tumor

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Verhaak et al. (2010). Cancer Cell

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Leek et al. (2010) Nat Reviews Genetics Batch effects in genomics data

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Processing*Batch* Biological*Group* Completely*confounded*study*design* Balanced*study*design* Rep:*1* Rep:*2* Rep:*2* Rep:*2* Rep:*1* Rep:*1* Group:*1* Group:*2* Group:*3* Batch:*3* Batch:*1* Batch:*2* Batch:*3* Observed*Differences* We*cannot*determine*if* variaCon*is*driven*by* biology*or*batch*effects* ● ● Batch Batch 1 Batch 2 Batch 3 ● ● Batch Batch 1 Batch 2 Batch 3 ● ● ● ● ● ● Batch Batch 1 Batch 2 Batch 3 Batch:*2* Batch:*1* Group:*3* Group:*1* Group:*2* The$Problem$of$Confounding$Biological$Varia6on$and$Batch$Effects$ ProporCon*of*detected*genes* ProporCon*of*detected*genes* Principal*Component*2* Principal*Component*2* Principal*Component*1* Principal*Component*1* Principal*Component*2* Principal*Component*1* ProporCon*of*detected*genes* Group*1* Group*1* Group*2* Group*3* Group*2* Group*3* Group*1* Group*2* Group*3* Good* Bad* When cells from one biological group or condition are cultured, captured and sequenced separate from cells in a second condition Confounded study design Batch effects in single-cell RNA-Seq data

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Processing*Batch* Biological*Group* Completely*confounded*study*design* Balanced*study*design* Rep:*1* Rep:*2* Rep:*2* Rep:*2* Rep:*1* Rep:*1* Group:*1* Group:*2* Group:*3* Batch:*3* Batch:*1* Batch:*2* Batch:*3* Observed*Differences* We*cannot*determine*if* variaCon*is*driven*by* biology*or*batch*effects* ● ● Batch Batch 1 Batch 2 Batch 3 ● ● Batch Batch 1 Batch 2 Batch 3 ● ● ● ● ● ● Batch Batch 1 Batch 2 Batch 3 Batch:*2* Batch:*1* Group:*3* Group:*1* Group:*2* The$Problem$of$Confounding$Biological$Varia6on$and$Batch$Effects$ ProporCon*of*detected*genes* ProporCon*of*detected*genes* Principal*Component*2* Principal*Component*2* Principal*Component*1* Principal*Component*1* Principal*Component*2* Principal*Component*1* ProporCon*of*detected*genes* Group*1* Group*1* Group*2* Group*3* Group*2* Group*3* Group*1* Group*2* Group*3* Good* Bad* When cells from one biological group or condition are cultured, captured and sequenced separate from cells in a second condition More balanced study design: Cells from different biological group processed in same batch Batch effects in single-cell RNA-Seq data

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Processing*Batch* Biological*Group* Completely*confounded*study*design* Balanced*study*design* Rep:*1* Rep:*2* Rep:*2* Rep:*2* Rep:*1* Rep:*1* Group:*1* Group:*2* Group:*3* Batch:*3* Batch:*1* Batch:*2* Batch:*3* Observed*Differences* We*cannot*determine*if* variaCon*is*driven*by* biology*or*batch*effects* ● ● Batch Batch 1 Batch 2 Batch 3 ● ● Batch Batch 1 Batch 2 Batch 3 ● ● ● ● ● ● Batch Batch 1 Batch 2 Batch 3 Batch:*2* Batch:*1* Group:*3* Group:*1* Group:*2* The$Problem$of$Confounding$Biological$Varia6on$and$Batch$Effects$ ProporCon*of*detected*genes* ProporCon*of*detected*genes* Principal*Component*2* Principal*Component*2* Principal*Component*1* Principal*Component*1* Principal*Component*2* Principal*Component*1* ProporCon*of*detected*genes* Group*1* Group*1* Group*2* Group*3* Group*2* Group*3* Group*1* Group*2* Group*3* Good* Bad* When cells from one biological group or condition are cultured, captured and sequenced separate from cells in a second condition More balanced study design: Cells from different biological group processed in same batch Batch effects in single-cell RNA-Seq data Proposed by Tung et al. (2016) bioRxiv

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Processing*Batch* Biological*Group* Completely*confounded*study*design* Balanced*study*design* Rep:*1* Rep:*2* Rep:*2* Rep:*2* Rep:*1* Rep:*1* Group:*1* Group:*2* Group:*3* Batch:*3* Batch:*1* Batch:*2* Batch:*3* Observed*Differences* We*cannot*determine*if* variaCon*is*driven*by* biology*or*batch*effects* ● ● Batch Batch 1 Batch 2 Batch 3 ● ● Batch Batch 1 Batch 2 Batch 3 ● ● ● ● ● ● Batch Batch 1 Batch 2 Batch 3 Batch:*2* Batch:*1* Group:*3* Group:*1* Group:*2* The$Problem$of$Confounding$Biological$Varia6on$and$Batch$Effects$ ProporCon*of*detected*genes* ProporCon*of*detected*genes* Principal*Component*2* Principal*Component*2* Principal*Component*1* Principal*Component*1* Principal*Component*2* Principal*Component*1* ProporCon*of*detected*genes* Group*1* Group*1* Group*2* Group*3* Group*2* Group*3* Group*1* Group*2* Group*3* Good* Bad* When cells from one biological group or condition are cultured, captured and sequenced separate from cells in a second condition More balanced study design: Cells from different biological group processed in same batch Batch effects in single-cell RNA-Seq data Implemented by Tung et al. (2016) bioRxiv

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Use FASTQ header as a surrogate for batch http://support.illumina.com/help/SequencingAnalysisWorkflow/Content/Vault/Informatics/Sequencing_Analysis/CASAVA/ swSEQ_mCA_FASTQFiles.htm

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Processing*Batch* Biological*Group* Completely*confounded*study*design* Balanced*study*design* Rep:*1* Rep:*2* Rep:*2* Rep:*2* Rep:*1* Rep:*1* Group:*1* Group:*2* Group:*3* Batch:*3* Batch:*1* Batch:*2* Batch:*3* Observed*Differences* We*cannot*determine*if* variaCon*is*driven*by* biology*or*batch*effects* ● ● Batch Batch 1 Batch 2 Batch 3 ● ● Batch Batch 1 Batch 2 Batch 3 ● ● ● ● ● ● Batch Batch 1 Batch 2 Batch 3 Batch:*2* Batch:*1* Group:*3* Group:*1* Group:*2* The$Problem$of$Confounding$Biological$Varia6on$and$Batch$Effects$ ProporCon*of*detected*genes* ProporCon*of*detected*genes* Principal*Component*2* Principal*Component*2* Principal*Component*1* Principal*Component*1* Principal*Component*2* Principal*Component*1* ProporCon*of*detected*genes* Group*1* Group*1* Group*2* Group*3* Group*2* Group*3* Group*1* Group*2* Group*3* Good* Bad* Hicks et al. (2015) bioRxiv

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Cells cluster by tumor Hicks et al. (2015) bioRxiv

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Cells cluster by batch (tumors are confounded with batch) Hicks et al. (2015) bioRxiv

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Hicks et al. (2015) bioRxiv

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Different batches have different detec=on rates Hicks et al. (2015) bioRxiv

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Deng et al. (2014) Science Treutlein et al. (2014) Nature Trapnell et al. (2014) Nat Biotech Patel et al. (2014) Science

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Batch and outcomes of interest are confounded in published scRNA-Seq experiments

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Differences in the detec=on rates between batches of cells Study design #1:

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Study design #2: Differences in the detec=on rates between batches of cells

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Two studies had completely confounded study designs

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Correla=on between PC1 and propor=on of detected genes Propor=on of detected genes Shalek et al. (2014). Nature Finak et al. (2015). Genome Biology

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