deconvo_lute-paper

Supplementary materials to prepare data and run analyses for the paper "lute: estimating the cell composition of heterogeneous tissue with varying cell sizes using gene expression" (1). The lute R/Bioconductor package (2) is publicly available for download.

Citation

Maden, Sean, Louise A. Huuki-Myers, Sang Ho Kwon, Leonardo Collado-Torres, Kristen R. Maynard, Stephanie C. Hicks. "lute: estimating the cell composition of heterogeneous tissue with varying cell sizes using gene expression" (manuscript in preparation)

Reproducing analyses

Scripts are provided in clearly-labeled folder. Script and folder names include numbers indicating their intended run order. Environment (.RData) and output (.rda) files may be generated by navigating one level above the scripts folder and entering the following from a shell prompt:

Rscript -e "source('PATH_TO_SCRIPT')"

or the following from an R session or script:

source('PATH_TO_SCRIPT')

Where PATH_TO_SCRIPT is the valid script path (e.g. ./scripts/02_pseudobulk/01_k2.R)

Figures and notebook builds are generated by building the .Rmd files in the notebooks folder.

Datasets

Folders huuki-meyers-et-al-2023_dlpfc-cohort1 (3,4), tran-et-al-2021_dlpfc-cohort2 (5), include data and analyses from neurotypical subject dorsolateral prefrontal cortex (DLPFC). Folder monaco-et-al-2019_pbmc-cohort1 (6) includes data and analyses of healthy subject peripheral blood mononuclear cell (PBMC) data.

Folders and directory structure

In addition to the dataset folders, software_manuscript includes additional files for the lute software vignettes and reporting RMSEs. dann-et-al-2023_env and sosina-et-al-2021_env include environment information from studies (7,8).

The file tree structure of this repo returned from tree -d:

├conda_env
├── dann-et-al-2023_env
│   └── yml
├── huuki-meyers-et-al-2023_dlpfc-cohort1
│   ├── data
│   │   └── 01_mae
│   ├── env
│   │   ├── 02_pseudobulk
│   │   ├── 02_summarize_mae
│   │   ├── 03_shuffle
│   │   ├── 04_experiment
│   │   └── 07_adjustment
│   ├── figures
│   │   ├── 02_pseudobulk
│   │   ├── 03_shuffle
│   │   └── 07_adjustment
│   ├── notebooks
│   │   ├── 00_preprocess
│   │   ├── 01_mae
│   │   ├── 02_pseudobulk
│   │   ├── 02_summarize_mae
│   │   ├── 03_shuffle
│   │   └── 07_adjustment
│   ├── outputs
│   │   ├── 00_preprocess
│   │   ├── 01_mae
│   │   ├── 02_pseudobulk
│   │   ├── 02_summarize_mae
│   │   ├── 03_shuffle
│   │   └── 07_adjustment
│   ├── scripts
│   │   ├── 00_preprocess
│   │   ├── 01_mae
│   │   ├── 02_pseudobulk
│   │   ├── 02_summarize_mae
│   │   ├── 03_shuffle
│   │   └── 07_adjustment
│   ├── source
│   └── unit_tests
│       ├── 02_mae_summaries
│       ├── 03_shuffle
│       └── 05_bulk
├── monaco-et-al-2019_pbmc-cohort1
│   ├── data
│   │   ├── cellScaleFactors
│   │   └── monaco_et_al_2019
│   │       ├── geo
│   │       ├── manuscript
│   │       │   └── original
│   │       └── shared
│   ├── env
│   │   └── 01_pseudobulk
│   ├── figures
│   │   └── 01_pseudobulk
│   ├── notebooks
│   │   └── 01_pseudobulk
│   │       └── 04_simulation_files
│   │           └── figure-latex
│   ├── outputs
│   │   └── 01_pseudobulk
│   ├── scripts
│   │   └── 01_pseudobulk
│   ├── source
│   └── yml
├── software_manuscript
│   ├── data
│   ├── env
│   │   └── 01_multipanel
│   ├── figures
│   │   ├── 01_multipanel
│   │   └── 02_diagram
│   ├── notebooks
│   │   ├── 01_multipanel
│   │   └── 02_aggregateRMSE
│   ├── outputs
│   │   └── 02_aggregateRMSE
│   ├── scripts
│   │   ├── 01_multipanel
│   │   ├── 02_diagram
│   │   └── 03_rmseTable
│   └── src
├── sosina-et-al-2021_env
│   ├── notebooks
│   ├── scripts
│   └── yml
└── tran-et-al-2021_dlpfc-cohort2
    ├── data
    ├── env
    │   └── 01_pseudobulk
    ├── figures
    │   └── 01_pseudobulk
    ├── notebooks
    │   └── 01_pseudobulk
    ├── outputs
    │   ├── 01_pseudobulk
    │   └── 02_summaries
    ├── scripts
    │   ├── 01_pseudobulk
    │   └── 02_summaries
    └── yml

The folder contents are described as follows:

huuki-meyers-et-al-2023_dlpfc-cohort1 : Materials for analyses of principal DLPFC dataset.
- ~/data : External data used by supplement.
- ~/env : Environment (.RData) saves from save.image().
- ~/figures : Figure saves from ~/notebooks.
- ~/notebooks : Notebooks (.Rmd) which load saved images and save figures.
- ~/outputs : Objects (.rda) saved by scripts.
- ~/scripts : Scripts (.Rmd) performing data preparation and analyses.
- ~/source : Scripts (.R) shared across analysis sections.
- ~/unit_tests : Scripts (.R) testing sample and sample source filters across analysis sections.
tran-et-al-2021_dlpfc-cohort2 : Materials for analyses of validation DLPFC dataset.
monaco-et-al-2019_pbmc-cohort1 : Materials for analyses of peripheral blood mononuclear cells (PBMC) dataset.
sosina-et-al-2021_env : Supplementary materials for analyses of previous work.
dann-et-al-2023_env : Supplementary materials for preprocessing samples for running with lute.

Manuscript Figures

Paths to manuscript figure sources.

Figure 1 : (see manuscript)
Figure 2A : huuki-meyers-et-al-2023_dlpfc-cohort1/notebooks/02_pseudobulk/01_k2.Rmd
Figure 2B : cohort3/notebooks/01_pseudobulk/04_simulation.Rmd"
Figure 3A : huuki-meyers-et-al-2023_dlpfc-cohort1/notebooks/07_adjustment/ 03_run_sopt_realbulk_all.Rmd
Figure 3B : huuki-meyers-et-al-2023_dlpfc-cohort1/notebooks/07_adjustment/03_run_sopt_realbulk_all.Rmd
Figure S1A : huuki-meyers-et-al-2023_dlpfc-cohort1/notebooks/02_pseudobulk/01_k2.Rmd
Figure S1B : tran-et-al-2021_dlpfc-cohort2/notebooks/01_pseudobulk/01_k2_mrb.Rmd
Figure S2A : huuki-meyers-et-al-2023_dlpfc-cohort1/notebooks/02_pseudobulk/02_k3.Rmd
Figure S2B : huuki-meyers-et-al-2023_dlpfc-cohort1/notebooks/02_pseudobulk/02_k3.Rmd
Figure S2C : tran-et-al-2021_dlpfc-cohort2/notebooks/01_pseudobulk/02_k3_mrb.Rmd
Figure S2D : huuki-meyers-et-al-2023_dlpfc-cohort1/notebooks/01_pseudobulk/02_k3_mrb.Rmd
Figure S3A : huuki-meyers-et-al-2023_dlpfc-cohort1/notebooks/03_shuffle/figS3A.Rmd
Figure S3B : huuki-meyers-et-al-2023_dlpfc-cohort1/notebooks/03_shuffle/figS3B.Rmd
Figure S4 : monaco-et-al-2019_pbmc-cohort1/notebooks/01_pseudobulk/04_simulations.Rmd"
Figure S5 : huuki-meyers-et-al-2023_dlpfc-cohort1/notebooks/07_adjustment/ 03_run_sopt_realbulk_all.Rmd
Figure S6 : huuki-meyers-et-al-2023_dlpfc-cohort1/notebooks/07_adjustment/03_run_sopt_realbulk_all.Rmd

Manuscript Tables

Paths to manuscript table sources.

Table S1 : huuki-meyers-et-al-2023_dlpfc-cohort1/notebooks/02_summarize_mae/TABLE_S1.csv
Table S2 : huuki-meyers-et-al-2023_dlpfc-cohort1/outputs/02_summarize_mae/TABLE_S2.csv
Table S3 : software/outputs/02_aggregateRMSE/TABLE_S3.csv
Table S4 : tran-et-al-2021_dlpfc-cohort2/outputs/02_summaries/TABLE_S4.csv
Table S5 : tran-et-al-2021_dlpfc-cohort2/outputs/02_summaries/cohort2_nuclei_summaries.csv
Table S6 : huuki-meyers-et-al-2023_dlpfc-cohort1/outputs/08_sizes/tables_cell_sizes.csv
Table S7 : huuki-meyers-et-al-2023_dlpfc-cohort1/outputs/03_shuffle/TABLE_S7.csv
Table S8 : huuki-meyers-et-al-2023_dlpfc-cohort1/outputs/02_summarize_mae/TABLE_S8.csv

Compute environment

Files to repeat analyses have been provided as .yml files in the ./deconvo_lute-paper/yml folder. These may be used to generate new conda environments. Additionally, top-level folders with .*_env in their name contain additional compute environment files for manuscript analyses.

Dataset descriptions

Single-nucleus RNA-seq (snRNA-seq) data

Single-nucleus RNA-seq (snRNA-seq) data from DLPFC were generated and preprocessed as described in Huuki-Meyers et al 2023. SingleCellExperiment files of these data are available from . Cell type labels for snRNA-seq datasets are determined from the variable cellType_broad_hc. This can be accessed from the sce object in various ways such as colData(sce)$cellType_broad_hc. Note that the deconvolution methods paper focuses on just 6 cell types of interest, and these are identified from among the cell type labels in the cellType_broad_hc variable.

Bulk RNA-seq data

The bulk RNA-seq datasets include 6 samples groups comprised of mRNA isolated from either nucleus, cytoplasm, or bulk, and prepped with either polyA selection or rRNA depletion strategies. Data come from 19 samples, meaning 113 total data points. Bulk RNA-seq data are available accessible as SummarizedExperiment files located at the project folder path:

Human_DLPFC_Deconvolution/processed-data/01_SPEAQeasy/

RNAscope data

Image data is generated for RNAscope slides by analysis with HALO and outputting the analysis results as .csv tables. These tables are read from the file tree located at the project folder path:

Human_DLPFC_Deconvolution/raw-data/HALO/Deconvolution_HALO_analysis/

Note, this contains one subdirectory for each of the RNAscope experiments, called Circle and Star, respectively. The processed data with confidence annotations files are located at the project folder path:

Human_DLPFC_Deconvolution/processed-data/03_HALO/halo_all.Rdata

Funding sources

1R01MH123183-01 (grantome_link)

Works cited

1. Maden, Sean, Louise A. Huuki-Myers, Sang Ho Kwon, Leonardo Collado-Torres, Kristen R. Maynard, Stephanie C. Hicks. "lute: estimating the cell composition of heterogeneous tissue with varying cell sizes using gene expression" (manuscript in preparation)

2. Maden, Sean K., Stephanie C. Hicks (2024). lute: Framework for cell size scale factor normalized bulk transcriptomics deconvolution experiments. doi:10.18129/B9.bioc.lute, R package version 0.99.25, DOI: 10.18129/B9.bioc.lute

3. Huuki-Myers, Louise, Abby Spangler, Nick Eagles, Kelsey D. Montgomery, Sang Ho Kwon, Boyi Guo, Melissa Grant-Peters, Heena R. Divecha, Madhavi Tippani, Chaichontat Sriworarat, Annie B. Nguyen, Prashanthi Ravichandran, Matthew N. Tran, Arta Seyedian, PsychENCODE consortium, Thomas M. Hyde, Joel E. Kleinman, Alexis Battle, Stephanie C. Page, Mina Ryten, Stephanie C. Hicks, Keri Martinowich, Leonardo Collado-Torres, and Kristen R. Maynard. 2023. “Integrated Single Cell and Unsupervised Spatial Transcriptomic Analysis Defines Molecular Anatomy of the Human Dorsolateral Prefrontal Cortex.” bioRxiv. https://doi.org/10.1101/2023.02.15.528722.

4. Huuki-Myers, Louise A., Kelsey D. Montgomery, Sang Ho Kwon, Stephanie Cinqueman, Sean K. Maden, Nicholas J. Eagles, Joel E. Kleinman, Thomas M. Hyde, Stephanie C. Hicks, Kristen R. Maynard, Leonardo Collado-Torres. "Benchmark of cellular deconvolution methods using a multi-assay reference dataset from postmortem human prefrontal cortex". BioRxiv. 2024 Feb 12. doi: https://doi.org/10.1101/2024.02.09.579665

5. Tran, Matthew N., Kristen R. Maynard, Abby Spangler, Louise A. Huuki, Kelsey D. Montgomery, Vijay Sadashivaiah, Madhavi Tippani, et al. 2021. “Single-Nucleus Transcriptome Analysis Reveals Cell-Type-Specific Molecular Signatures across Reward Circuitry in the Human Brain.” Neuron 109 (19): 3088-3103.e5. https://doi.org/10.1016/j.neuron.2021.09.001.

6. Monaco, Gianni, Bernett Lee, Weili Xu, Seri Mustafah, You Yi Hwang, Christophe Carré, Nicolas Burdin, Lucian Visan, Michele Ceccarelli, Michael Poidinger, Alfred Zippelius, João Pedro de Magalhães, Anis Larbi. 2019. "RNA-Seq Signatures Normalized by mRNA Abundance Allow Absolute Deconvolution of Human Immune Cell Types." Cell Reports 26 (6): 1627-1640.e7. https://doi.org/10.1016/j.celrep.2019.01.041.

7. Sosina, Olukayode A., Matthew N. Tran, Kristen R. Maynard, Ran Tao, Margaret A. Taub, Keri Martinowich, Stephen A. Semick, Bryan C. Quach, Daniel R. Weinberger, Thomas Hyde, Dana B. Hancock, Joel E. Kleinman, Jeffrey T. Leek, Andrew E. Jaffe. Strategies for cellular deconvolution in human brain RNA sequencing data. F1000Res. 2021 Aug 4;10:750. doi.org/10.12688/f1000research.50858.1

8. Dann, Emma, Ana-Maria Cujba, Amanda J. Oliver, Kerstin B. Meyer, Sarah A. Teichmann, and John C. Marioni. 2023. “Precise Identification of Cell States Altered in Disease Using Healthy Single-Cell References.” Nature Genetics 55 (11): 1998–2008. https://doi.org/10.1038/s41588-023-01523-7.

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