Welcome to RCTD, an R package for assigning cell types to spatial transcriptomics data. RCTD inputs a spatial transcriptomics dataset, which consists of a set of pixels, which are spatial locations that measure RNA counts across many genes. RCTD additionally uses a single cell RNA-seq (scRNA-seq) dataset, which is labeled for cell types. RCTD learns cell type profiles from the scRNA-seq dataset, and uses these to label the spatial transcriptomics pixels as cell types. Notably, RCTD allows for individual pixels to be cell type mixtures; that is, they can potentially source RNA from multiple cell types. RCTD identifies the cell types on each pixel, and estimates the proportion of each of these cell types. Additionally, RCTD has a platform effect normalization step, which normalizes the scRNA-seq cell type profiles to match the platform effects of the spatial transcriptomics dataset. A platform effect is the tendency of a sequencing technology to capture individual genes at different rates.

Code for generating the figures of our paper, Robust decomposition of cell type mixtures in spatial transcriptomics, is located here. Our paper can be found here.

You can install the current version of RCTD from GitHub with:

# install.packages("devtools")
devtools::install_github("dmcable/RCTD", build_vignettes = TRUE)

To subsequently view the vignette (recommended for learning how to use RCTD):

browseVignettes('RCTD')

In this section, we aim to explain how to use RCTD as quickly as possible on your data:

1. Open the ‘spatial-transcriptomics.Rmd’ vignette for a complete explanation of the RCTD workflow. Expected output of the vignette is provided here.

2. As described in the ‘Data Preprocessing’ step of the vignette, convert your spatial transcriptomics data to a SpatialRNA object (called puck here) and your scRNA-seq reference to a Seurat object (called reference here).

3. Run RCTD. You can optionally set test_mode to TRUE in create.RCTD to quickly test RCTD, but you should set it to FALSE for the official run.

myRCTD <- create.RCTD(puck, reference, max_cores = 8, test_mode = FALSE) # here puck is the SpatialRNA object, and reference is the Seurat object.
myRCTD <- run.RCTD(myRCTD, doublet_mode = TRUE)

4. Observe RCTD results. RCTD results are stored in the @results field. Of particular interest is @results$weights, a data frame of cell type weights for each pixel (for full mode). This section will generate various plots which can be found in resultsdir. The results of ‘doublet_mode’ are stored in @results$results_df and @results$weights_doublet, the weights of each cell type. More specifically, the results_df object contains one column per pixel (barcodes as rownames). Important columns are:

• spot_class, a factor variable representing RCTD’s classification in doublet mode: “singlet” (1 cell type on pixel), “doublet_certain” (2 cell types on pixel), “doublet_uncertain” (2 cell types on pixel, but only confident of 1), “reject” (no prediction given for pixel).
• Next, the first_type column gives the first cell type predicted on the bead (for all spot_class conditions except “reject”).
• The second_type column gives the second cell type predicted on the bead for doublet spot_class conditions (not a confident prediction for “doublet_uncertain”).

For some example of summary plots, follow the ‘RCTD results’ section of the ‘spatial-transcriptomics’ vignette.

• R version >= 3.5.0.
• R packages: readr, Seurat, pals, ggplot2, Matrix, doParallel, foreach, quadprog, tibble, dplyr, reshape2.

For optimal performance, we recommend at least 4 GB of RAM, and multiple cores may be used to speed up runtime.

Installation time: Less than five minutes, after installing dependent packages. RCTD takes up approximately 145 MB of space due to pre-computed data tables that significantly improve performance.

Runtime: The example dataset provided (Vignette) can be run in less than 1 minutes on a normal desktop computer. Approximately 20 minutes on a Slide-seq cerebellum dataset (approximately 3,000 genes and 11,000 pixels) on a laptop computer with 8 cores. Using less cores will lead to longer runtime.

Operating systems (version 1.1.0 RCTD) tested on:

• macOS Mojave 10.14.6
• GNU/Linux (GNU coreutils) 8.22

RCTD is licensed under the GNU General Public License v3.0.