Towards progress in batch effects and biases in single-cell RNA-Seq data

Presented at the 2016 Single Cell Genomics Conference at the Wellcome Genome Campus in Hinxton, UK

1. 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
2. Bimodal distribu=on of expression Number of Cells Expression level of Gene X μ 1 μ 2 True Expression
3. Bimodal distribu=on of expression Expression level of Gene X μ 1 μ 2 True Expression Observed Expression Number of Cells
4. Patel et al. (2014) Science Cells cluster by tumor
5. Verhaak et al. (2010). Cancer Cell
6. Leek et al. (2010) Nat Reviews Genetics Batch effects in genomics data
7. 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
8. 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
9. 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
10. 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
11. Use FASTQ header as a surrogate for batch http://support.illumina.com/help/SequencingAnalysisWorkflow/Content/Vault/Informatics/Sequencing_Analysis/CASAVA/ swSEQ_mCA_FASTQFiles.htm
12. 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
13. Cells cluster by tumor Hicks et al. (2015) bioRxiv
14. Cells cluster by batch (tumors are confounded with batch) Hicks et al. (2015) bioRxiv
15. Hicks et al. (2015) bioRxiv
16. Different batches have different detec=on rates Hicks et al. (2015) bioRxiv
17. Deng et al. (2014) Science Treutlein et al. (2014) Nature Trapnell et al. (2014) Nat Biotech Patel et al. (2014) Science
18. Batch and outcomes of interest are confounded in published scRNA-Seq experiments
19. Differences in the detec=on rates between batches of cells Study design #1:
20. Study design #2: Differences in the detec=on rates between batches of cells
21. Two studies had completely confounded study designs
22. 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