Single cells, simulation and kidneys in a dish

Single-cell RNA sequencing (scRNA-seq) is rapidly becoming a tool of choice for biologists wishing to investigate gene expression at greater resolution, particularly in areas such as development and differentiation. Single-cell data presents an array of bioinformatics challenges, data is sparse (for both biological and technical reasons), quality control is difficult and it is unclear how to replicate measurements. As scRNA-seq datasets have become available so have a plethora of analysis methods. We have catalogued software tools that implement these methods in the scRNA-tools database (www.scRNA-tools.org). Evaluation of analysis methods relies on having a truth to test against or deep biological knowledge to interpret the results. Unfortunately current scRNA-seq simulations are frequently poorly documented, not reproducible and do not demonstrate similarity to real data or experimental designs. In this talk I will present Splatter, a Bioconductor package for simulating scRNA-seq data that is designed to address these issues. Splatter provides a consistent, easy to use interface to several previously published simulations allowing researchers to estimate parameters, produce synthetic datasets and compare how well they replicate real data. Splatter also includes Splat, our own simulation model. Based on a gamma-Poisson hierarchical model, Splat includes additional features often seen in scRNA-Seq data, such as dropout, and can be used to simulate complex experiments including multiple cell types, differentiation lineages and multiple batches. I will also briefly discuss an analysis of a complex kidney organoid dataset, showing how more cells and different levels of clustering help to reveal greater biological insight.

Transcript

1. Single-cells,
   simulation and
   kidneys in a dish
   Luke Zappia
   MCRI Bioinformatics
   @_lazappi_
   

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3. Parkville Precinct
   MCRI
   

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4. MCRI Bioinformatics
   bpipe Corset
   Lace
   Necklace
   GOseq
   Splatter
   clinker
   JAFFA
   Cpipe
   Ximmer
   Schism
   missMethyl
   STRetch
   Structural
   Clinical
   STRs
   Single-cell
   Pipelines
   Gene sets
   Fusions
   Assembly
   superTranscripts
   Methylation
   scRNA-tools
   

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5. Bulk RNA-seq
   ACTGACTCCA
   TCAGTACTGA
   CGTGTCATAG
   GATTGACCTA
   Gene Sample 1
   A 43
   B 3
   C 17
   D 24
   

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6. Single-cell RNA-seq
   ACTGACTCCA
   TCAGTACTGA
   CGTGTCATAG
   GATTGACCTA
   ACTGACTCCA
   TCAGTACTGA
   CGTGTCATAG
   GATTGACCTA
   ACTGACTCCA
   TCAGTACTGA
   CGTGTCATAG
   GATTGACCTA
   ACTGACTCCA
   TCAGTACTGA
   CGTGTCATAG
   GATTGACCTA
   Gene Cell 1 Cell 2 Cell 3 Cell 4
   A 12 10 9 0
   B 0 0 0 1
   C 9 6 0 0
   D 7 0 4 0
   

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7. Moore’s Law
   Sevensson et al. arXiv 1704.01379, 2017
   

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9. Unique Molecular Identifiers
   UMIs
   5’
   3’
   AAAA
   (PCR){BC}[UMI]TTTT
   5 4
   Aligned reads De-duplication and counting
   

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10. Gene Cell 1 Cell 2 Cell 3 Cell 4
    A 12 10 9 0
    B 0 0 0 1
    C 9 6 0 0
    D 7 0 4 0
    

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11. Gene Cell 1 Cell 2 Cell 3 Cell 4
    A 12 0 10 9 0
    B 0 0 0 1
    C 9 6 0 0
    D 7 0 4 0
    Bad cell?
    Low expression?
    Cell type specific?
    Cell cycle?
    Dropout?
    

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15. www. .org
    

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19. Simulation
    Biology
    Evaluation
    

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20. Simulation
    

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21. Simulations
    Provide a truth to test against
    BUT
    - Often poorly documented and explained
    - Not easily reproducible or reusable
    - Don’t demonstrate similarity to real data
    

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22. “Splatter: simulation of single-cell RNA sequencing data.”
    Genome Biology (2017) DOI: 10.1186/s13059-017-1305-0
    

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23. Splatter
    Bioconductor package
    Collection of simulation methods
    Consistent, easy to use, interface
    Functions for comparison
    

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24. Negative binomial
    

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25. Splat
    Negative binomial
    Expression outliers
    Defined library sizes
    Mean-variance trend
    Dropout
    

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26. Simple - Negative binomial
    Lun - NB with cell factors
    Lun ATL, Bach K, Marioni JC. Genome Biology (2016).
    DOI: 10.1186/s13059-016-0947-7.
    Lun 2 - Sampled NB with batch effects
    Lun ATL, Marioni JC. Biostatistics (2017).
    DOI: 10.1093/biostatistics/kxw055.
    Simulations
    scDD - NB with bimodality
    Korthauer KD, et al. Genome Biology (2016).
    DOI: 10.1186/s13059-016-1077-y.
    BASiCS - NB with spike-ins
    Vallejos CA, Marioni JC, Richardson S. PLoS Comp. Bio. (2015).
    DOI: 10.1371/journal.pcbi.1004333.
    

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27. Using Splatter
    params1 params2 sim1 sim2 datasets Splat = sim1,
    Simple = sim2)
    comp diff 1. Estimate
    2. Simulate
    3. Compare
    

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28. Real data
    3 HapMap individuals
    3 plates each
    200 random cells
    Tung P-Y et al. Sci. Rep. (2017) DOI:10.1038/srep39921
    A1 A2 A3
    A
    B1 B2 B3
    B
    C1 C2 C3
    C
    Tung et al. iPSCs, C1 capture
    

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29. Means Difference in Means
    

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30. Zeros per cell Difference in zeros
    

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31. Mean-zeros Difference
    

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32. Rank 1 8
    

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33. Rank 1 8
    Full-length
    Full-length
    

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34. Complex simulations
    Groups Batches Paths
    

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35. Example evaluation
    Parameters
    - Estimated from Tung data
    Simulation
    - 400 cells
    - 3 groups (60%, 25%, 15%)
    - 10% DE (~1700 genes)
    - 20 replicates
    Method
    - SC3
    - k-means consensus clustering
    - Differential expression
    - Marker genes
    

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36. Clustering Gene identification
    

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37. New in Splatter 1.2.0
    SingleCellExperiment
    Batch effects
    Simulations
    - BASiCS
    - mfa
    - PhenoPath
    - ZINB-WaVE
    Bioconductor 3.6
    

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38. Simulation summary
    Simulations are a great tool
    But they should be:
    - Reusable
    - Reproducible
    - Realistic
    Splatter is our solution Genome Biology
    10.1186/s13059-017-1305-0
    

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39. Biology
    

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40. The kidney
    OpenStax College, CC BY 3.0 via Wikimedia Commons
    

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41. Organoids
    Day 0 4
    7 10 18 25
    CHIR FGF9
    FGF9
    CHIR
    Form pellets
    No GF
    iPSCs organoid
    Takasato M et al. Nature. (2015) DOI: 10.1038/nature15695
    

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42. GATA3
    ECAD
    LTL
    WT1
    CD +
    DT +
    PT +
    Glo
    

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43. Fluidigm experiment
    4 organoids
    C1 capture
    Full-length
    No spike-ins
    

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44. Analysis
    Alignment
    Quantification
    Quality control
    Clustering
    Gene detection
    Interpretation
    STAR
    featureCounts
    scater
    SC3
    SC3
    Biologists
    

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45. Quality control
    Cells
    - Alignment
    - Quantification
    - Expression
    278 -> 155
    Genes
    - Expression
    - Class
    23388
    

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46. Clustering
    

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47. 10x experiment
    3 organoids
    Chromium capture
    UMI
    ~7000 cells
    

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48. Analysis
    CellRanger
    CellRanger
    scater
    Seurat
    Seurat
    Biologists
    Alignment
    Quantification
    Quality control
    Clustering
    Gene detection
    Interpretation
    

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49. Three clusters
    Vasculature
    Epithelium
    “Stroma”
    

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50. Many clusters
    

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51. Vasculature
    Proximal tubule
    Podocytes
    

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52. Mesangium Renal stroma
    

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53. Nephron?
    Neuronal?
    

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54. ?
    

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56. Nodes
    Resolution (k)
    Cluster
    Size
    

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57. Edges
    Cluster from
    (lower resolution)
    Cluster to
    (higher resolution)
    Number
    Proportion
    

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58. Proportions
    100
    60
    40
    k = 1 k = 2
    p
    from
    = n / size
    low
    n = 60
    n = 40
    p
    to
    = n / size
    high
    

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59. Clustering tree
    Resolution
    0.01
    0.1
    0.2
    0.3
    0.4
    0.5
    0.6
    0.7
    0.8
    0.9
    1.0
    

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60. Resolution
    0.01
    0.1
    0.2
    0.3
    0.4
    0.5
    0.6
    0.7
    0.8
    0.9
    1.0
    

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61. Resolution
    0.01
    0.1
    0.2
    0.3
    0.4
    0.5
    0.6
    0.7
    0.8
    0.9
    1.0
    Vasculature
    

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62. Resolution
    0.01
    0.1
    0.2
    0.3
    0.4
    0.5
    0.6
    0.7
    0.8
    0.9
    1.0
    Proximal
    Tubule
    Podocytes
    

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63. Resolution
    0.01
    0.1
    0.2
    0.3
    0.4
    0.5
    0.6
    0.7
    0.8
    0.9
    1.0
    Mesangium
    

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64. Resolution
    0.01
    0.1
    0.2
    0.3
    0.4
    0.5
    0.6
    0.7
    0.8
    0.9
    1.0
    Renal stroma
    

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65. Resolution
    0.01
    0.1
    0.2
    0.3
    0.4
    0.5
    0.6
    0.7
    0.8
    0.9
    1.0
    Nephron/neuronal?
    

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66. Resolution
    0.01
    0.1
    0.2
    0.3
    0.4
    0.5
    0.6
    0.7
    0.8
    0.9
    1.0
    ?
    

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67. Summary
    Kidney organoids are complex
    More cells help
    Cluster relationships can be useful
    Background knowledge is vital
    

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68. Acknowledgements
    Everyone that makes tools and data available
    Supervisors
    Alicia Oshlack
    Melissa Little
    MCRI Bioinformatics
    Belinda Phipson
    Breon Schmidt
    MCRI KDDR
    Alex Combes
    

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69. @_lazappi_
    oshlacklab.com
    www.scRNA-tools.org
    @scRNAtools
    “Splatter: simulation of
    single-cell RNA sequencing
    data.”
    Genome Biology (2017)
    DOI:
    10.1186/s13059-017-1305-0
    tinyurl.com/clust-tree-funcs
    “Exploring the single-cell
    RNA-seq analysis landscape
    with the scRNA-tools database”
    bioRxiv (2017)
    DOI: 10.1101/206573
    bioconductor.org/packages/
    splatter
    

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