# Using Monocle as input to tradeSeq

# Introduction

`tradeSeq` is an `R` package that allows analysis of gene expression along trajectories. While it has been developed and applied to single-cell RNA-sequencing (scRNA-seq) data, its applicability extends beyond that, and also allows the analysis of, e.g., single-cell ATAC-seq data along trajectories or bulk RNA-seq time series datasets. For every gene in the dataset, `tradeSeq` fits a generalized additive model (GAM) by building on the `mgcv` R package. It then allows statistical inference on the GAM by assessing contrasts of the parameters of the fitted GAM model, aiding in interpreting complex datasets. All details about the `tradeSeq` model and statistical tests are described in our preprint (Van den Berge et al. 2020).

In this vignette, we analyze a subset of the data from (Paul et al. 2015). A `SingleCellExperiment` object of the data has been provided with the `tradeSeq` package and can be retrieved as shown below. The data and UMAP reduced dimensions were derived from following the Monocle 3 vignette.

The main vignette focuses on using `tradeSeq` downstream of `slingshot`. Here, we present how to use `tradeSeq` downstream of `monocle`(Qiu et al. 2017).

# Load data

    library(tradeSeq)
    library(RColorBrewer)
    library(SingleCellExperiment)

    # For reproducibility
    palette(brewer.pal(8, "Dark2"))
    data(countMatrix, package = "tradeSeq")
    counts <- as.matrix(countMatrix)
    rm(countMatrix)
    data(celltype, package = "tradeSeq")

# Monocle3

As of now (06/2020), monocle3(Cao et al. 2019), is still in its beta version. Therefore, we have no plan yet to include a S4 method for monocle3 while it is not on CRAN or Bioconductor and the format is still moving. However, we present below a way to use `tradeSeq` downstream of `monocle3` as of version ‘0.2’, for a fully connected graph. We follow the tutorial from the monocle3 website.

## Constructing the trajectory

You will need to install monocle3 from here before running the code below.

    set.seed(22)
    library(monocle3)
    # Create a cell_data_set object
    cds <- new_cell_data_set(counts, cell_metadata = pd,
                    gene_metadata = data.frame(gene_short_name = rownames(counts),
                                               row.names = rownames(counts)))
    # Run PCA then UMAP on the data
    cds <- preprocess_cds(cds, method = "PCA")
    cds <- reduce_dimension(cds, preprocess_method = "PCA",
                            reduction_method = "UMAP")

    # First display, coloring by the cell types from Paul et al
    plot_cells(cds, label_groups_by_cluster = FALSE, cell_size = 1,
               color_cells_by = "cellType")

    # Running the clustering method. This is necessary to the construct the graph
    cds <- cluster_cells(cds, reduction_method = "UMAP")
    # Visualize the clusters
    plot_cells(cds, color_cells_by = "cluster", cell_size = 1)

    # Construct the graph
    # Note that, for the rest of the code to run, the graph should be fully connected
    cds <- learn_graph(cds, use_partition = FALSE)

    # We find all the cells that are close to the starting point
    cell_ids <- colnames(cds)[pd$cellType ==  "Multipotent progenitors"]
    closest_vertex <- cds@principal_graph_aux[["UMAP"]]$pr_graph_cell_proj_closest_vertex
    closest_vertex <- as.matrix(closest_vertex[colnames(cds), ])
    closest_vertex <- closest_vertex[cell_ids, ]
    closest_vertex <- as.numeric(names(which.max(table(closest_vertex))))
    mst <- principal_graph(cds)$UMAP
    root_pr_nodes <- igraph::V(mst)$name[closest_vertex]

    # We compute the trajectory
    cds <- order_cells(cds, root_pr_nodes = root_pr_nodes)
    plot_cells(cds, color_cells_by = "pseudotime")

## Extracting the pseudotimes and cell weights for tradeSeq

    library(magrittr)
    # Get the closest vertice for every cell
    y_to_cells <-  principal_graph_aux(cds)$UMAP$pr_graph_cell_proj_closest_vertex %>%
      as.data.frame()
    y_to_cells$cells <- rownames(y_to_cells)
    y_to_cells$Y <- y_to_cells$V1

    # Get the root vertices
    # It is the same node as above
    root <- cds@principal_graph_aux$UMAP$root_pr_nodes

    # Get the other endpoints
    endpoints <- names(which(igraph::degree(mst) == 1))
    endpoints <- endpoints[!endpoints %in% root]

    # For each endpoint
    cellWeights <- lapply(endpoints, function(endpoint) {
      # We find the path between the endpoint and the root
      path <- igraph::shortest_paths(mst, root, endpoint)$vpath[[1]]
      path <- as.character(path)
      # We find the cells that map along that path
      df <- y_to_cells[y_to_cells$Y %in% path, ]
      df <- data.frame(weights = as.numeric(colnames(cds) %in% df$cells))
      colnames(df) <- endpoint
      return(df)
      }) %>% do.call(what = 'cbind', args = .) %>%
        as.matrix()
    rownames(cellWeights) <- colnames(cds)
    pseudotime <- matrix(pseudotime(cds), ncol = ncol(cellWeights),
                         nrow = ncol(cds), byrow = FALSE)

    sce <- fitGAM(counts = counts,
                  pseudotime = pseudotime,
                  cellWeights = cellWeights)

Then, the `sce` object can be analyzed following the main vignette.

# Session

    sessionInfo()

    ## R version 4.6.0 (2026-04-24)
    ## Platform: x86_64-pc-linux-gnu
    ## Running under: Ubuntu 24.04.4 LTS
    ##
    ## Matrix products: default
    ## BLAS:   /usr/lib/x86_64-linux-gnu/openblas-pthread/libblas.so.3
    ## LAPACK: /usr/lib/x86_64-linux-gnu/openblas-pthread/libopenblasp-r0.3.26.so;  LAPACK version 3.12.0
    ##
    ## Random number generation:
    ##  RNG:     Mersenne-Twister
    ##  Normal:  Inversion
    ##  Sample:  Rounding
    ##
    ## locale:
    ##  [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C
    ##  [3] LC_TIME=en_US.UTF-8        LC_COLLATE=en_US.UTF-8
    ##  [5] LC_MONETARY=en_US.UTF-8    LC_MESSAGES=en_US.UTF-8
    ##  [7] LC_PAPER=en_US.UTF-8       LC_NAME=C
    ##  [9] LC_ADDRESS=C               LC_TELEPHONE=C
    ## [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C
    ##
    ## time zone: Etc/UTC
    ## tzcode source: system (glibc)
    ##
    ## attached base packages:
    ## [1] stats4    stats     graphics  grDevices utils     datasets  methods
    ## [8] base
    ##
    ## other attached packages:
    ##  [1] mgcv_1.9-4                  nlme_3.1-169
    ##  [3] slingshot_2.21.0            TrajectoryUtils_1.21.0
    ##  [5] princurve_2.1.6             SingleCellExperiment_1.35.1
    ##  [7] SummarizedExperiment_1.43.0 Biobase_2.73.1
    ##  [9] GenomicRanges_1.65.0        Seqinfo_1.3.0
    ## [11] IRanges_2.47.1              S4Vectors_0.51.3
    ## [13] BiocGenerics_0.59.6         generics_0.1.4
    ## [15] MatrixGenerics_1.25.0       matrixStats_1.5.0
    ## [17] RColorBrewer_1.1-3          tradeSeq_1.27.0
    ## [19] knitr_1.51                  rmarkdown_2.31
    ##
    ## loaded via a namespace (and not attached):
    ##  [1] gtable_0.3.6        xfun_0.57           bslib_0.11.0
    ##  [4] ggplot2_4.0.3       lattice_0.22-9      vctrs_0.7.3
    ##  [7] tools_4.6.0         parallel_4.6.0      tibble_3.3.1
    ## [10] pkgconfig_2.0.3     Matrix_1.7-5        S7_0.2.2
    ## [13] lifecycle_1.0.5     compiler_4.6.0      farver_2.1.2
    ## [16] statmod_1.5.2       codetools_0.2-20    htmltools_0.5.9
    ## [19] sys_3.4.3           buildtools_1.0.0    sass_0.4.10
    ## [22] yaml_2.3.12         pillar_1.11.1       jquerylib_0.1.4
    ## [25] BiocParallel_1.47.0 limma_3.69.1        DelayedArray_0.39.3
    ## [28] cachem_1.1.0        viridis_0.6.5       abind_1.4-8
    ## [31] locfit_1.5-9.12     tidyselect_1.2.1    digest_0.6.39
    ## [34] dplyr_1.2.1         labeling_0.4.3      maketools_1.3.2
    ## [37] splines_4.6.0       fastmap_1.2.0       grid_4.6.0
    ## [40] cli_3.6.6           SparseArray_1.13.2  magrittr_2.0.5
    ## [43] S4Arrays_1.13.0     withr_3.0.2         edgeR_4.11.1
    ## [46] scales_1.4.0        XVector_0.53.0      igraph_2.3.1
    ## [49] gridExtra_2.3       pbapply_1.7-4       evaluate_1.0.5
    ## [52] viridisLite_0.4.3   rlang_1.2.0         Rcpp_1.1.1-1.1
    ## [55] glue_1.8.1          jsonlite_2.0.0      R6_2.6.1

# References

Cao, Junyue, Malte Spielmann, Xiaojie Qiu, et al. 2019. “The Dynamics and Regulators of Cell Fate Decisions Are Revealed by Pseudo-Temporal Ordering of Single Cells.” Nature.
Paul, Franziska, Ya’ara Arkin, Amir Giladi, et al. 2015. “Transcriptional Heterogeneity and Lineage Commitment in Myeloid Progenitors.” Cell 163 (7): 1663–77. https://doi.org/10.1016/J.CELL.2015.11.013.
Qiu, Xiaojie, Qi Mao, Ying Tang, et al. 2017. “Reversed graph embedding resolves complex single-cell trajectories.” Nature Methods 14 (10): 979–82. https://doi.org/10.1038/nmeth.4402.
Van den Berge, Koen, Hector Roux de Bézieux, Kelly Street, et al. 2020. “Trajectory-based differential expression analysis for single-cell sequencing data.” Nature Communications 11 (1): 1201. https://doi.org/10.1038/s41467-020-14766-3.