NB-FOXR2
Analysis for "FOXR2 targets LHX6+/DLX+ neural lineages to drive CNS neuroblastoma" (Jessa*, De Cola*, Chandarana*, et al, Cancer Research, 2024)
NB-FOXR2 analysis code
- This is the public repository accompanying the study: FOXR2 targets LHX6+/DLX+ neural lineages to drive CNS neuroblastoma, Jessa*, De Cola*, Chandarana*, …, Pathania, Jabado, Kleinman, Cancer Research, 2024 (link)
- Link to repository: https://github.com/fungenomics/NB-FOXR2
- This repository is archived on Zenodo
- This repository contains primarily code, see the Data availability section for links to data
- See the Code to reproduce analyses section for links to code of each figure and table
Contents of this repository
Codebase overview
- This repository is meant to enhance the Materials & Methods section by providing code for the custom analyses in the manuscript and the exact R dependencies, in order to improve reproducibility for the main results. However, it is not a fully executable workflow.
- In general, read alignment and cell calling for single-cell sequencing data has been performed using in-house pipelines, not included here. This repository mainly contains custom/downstream code.
- A first level of downstream analysis involves scripts applied in parallel to individual samples for specific data types.
Copies of these scripts are provided in the
code/scripts/per_sample_script_examples(the execution of these scripts is performed in thedata/singlecell,data/RNAseq, etc folders, not included here). - A second level of downstream analysis involves custom analyses, aggregating samples and data types, use to derive the main results included in the paper. These are provided in .Rmd files in
code, with the associated .md and rendered HTML files.
Codebase structure
renv.lock–> lockfile containing all package versions for R analysiscode–> code for R analysis, contains the .Rmd files that run the high-level analyses and produce figures included in the paperfunctions–> contains .R files with custom functions used throughout the analysisscripts–> contains .R and bash scripts for analyses that are repeated on individual samples, as well as helper scripts e.g. for formatting data
include–> contains templates, palettes, etc, for this repositoryrr_helpers.R–> contains helper functions for working with this GitHub repository template (rr)
Analysis documents
Rendered HTML files for each analysis can be viewed here:
- 01: Oncoprint
- 02: Prepare reference gene signatures
- 03: Process human tumor bulk RNAseq
- 04: Process human tumor single-cell RNAseq
- 05: Transcription factor fingerprint
- 06.1: Project bulk human tumors to normal references
- 06.2: Project single-cell human tumors to normal references
- 07.1: Project single-cell mouse models to normal references
- 07.2: Validate similarity of mouse models to human disease
- 08: Compare to extracranial neuroblastoma
- 09: Process and analyse FOXR2 CUT&RUN chromatin binding
Code to reproduce analyses
Code to reproduce analyses is saved in code/.
Figures
Source of each Main Figure in the R markdowns:
| Figure number | Description | R markdown |
|---|---|---|
| 1a | Oncoprint | 01 |
| 1b | FOXR2 expression in bulk human brain tumors | 03 |
| 2a | TF fingerprint expression in normal brain | NA - Custom schematic + imaging from online browsers (link, link) |
| 2b-c | TF fingerprint SVM and expression in normal development | 05 |
| 3a, c-d | TF fingerprint expression in selected bulk human tumors | 05 |
| 3b | Comparison of TF expression in bulk FOXR2+/- extracranial neuroblastoma (Gartlgruber 2021 dataset) | 08 |
| 4a-b, e-f | ssGSEA scoring in bulk human tumors | 06.1 |
| 4c (top), 4d | Single-cell NB-FOXR2 mapping | 06.2 |
| 4c (bottom) | Single-cell NB-FOXR2 colored by sample | 04 |
| 5 | Survival and imaging of murine models | NA - figures from Pathania lab |
| 6a-b | Single-cell murine models inferCNV and UMAP colored by broad cell classes | 07.1 |
| 6c-f | Human tumor signature generation and ssGSEA scoring in murine models (neuron-like cells) | 07.2 |
| 7a-c | Mouse model qPCR and survival curve | NA - figures from Pathania lab |
| 7d-e | FOXR2 CUT&RUN peak overlap and motif enrichment | 09 |
| 7f-i | Genomic tracks of CUT&RUN, ATAC, RNAseq | NA - figures from IGV |
Source of each Supplementary Figure in the R markdowns:
| Supplementary figure number | Title | R markdown |
|---|---|---|
| 1a-b | Single-cell tumor QC | 04 |
| 1c | inferCNV in human tumors | NA - png files from data/singlecell/pipeline_sc*/{sample}/inferCNV |
| 1d | MDM4 expression in human bulk tumors | 03 |
| 1e | MDM4 expression in human single-cell tumors | 06.2 |
| 2 | TF fingerprint expression in individual clusters of normal development | 05 |
| 3a-b | TF fingerprint expression in all human bulk tumors | 05 |
| 3c | Comparison of TF expression in bulk FOXR2+/- extracranial neuroblastoma (TARGET dataset) | 08 |
| 4 | Top ssGSEA scoring signatures per human bulk brain tumor, indiscriminant signature (see Methods) | 06.1 |
| 5a | OL marker expression in human bulk brain tumors | 05 |
| 5b | Expression of neuronal markers in normal brain | NA - from online browser |
| 5c | Neuronal marker expression in human single-cell NB-FOXR2 | 06.2 |
| 6a, c-d | Single-cell murine models cell type annotation and marker expression | 07.1 |
| 6b | inferCNV in malignant cells of murine models | NA - png files from data/singlecell/mm_inferCNV/{sample}/malignant_only |
| 6e | Human tumor signature scoring in glial-like cells of murine models | 07.2 |
| 7 | Imaging of orthotopic engraftment | NA - figures from Pathania lab |
Tables
Source of each Supplementary Table in the R markdowns (denoted by section titled TABLE...):
| Supplementary table number | Title | R markdown |
|---|---|---|
| 1 | Murine models | NA – murine experimental information from Pathania lab |
| 2 | NGS summary | NA - generated external to this repo |
| 3 | Human tumor bulk QC | 03 |
| 4 | Human tumor singlecell QC | 04 |
| 5 | Murine models singlecell QC | 07.1 |
| 6 | Normal reference datasets | NA – created manually, collects references used in 02, 05, 06.2, 07.1 |
| 7 | Murine models annotation reference labels | 07.1 |
| 8 | TF quantification | 05 |
| 9 | Reference gene signatures | 06.1 |
| 10 | Cell type signature enrichment | 06.1 |
Data availability
Data from this study
- Raw and processed data for murine samples have been deposited to GEO at GSE270666.
- Raw data for human tumors have been deposited to EGA at EGAS00001007247.
- For human samples, please see the associated Zenodo record for processed data including counts matrices, cell annotations, and bigWig files.
External data
-
Gartlgruber 2021 dataset of extracranial neuroblastoma (bulk RNA-seq) analyzed in this study were obtained as count matrices from a public Shiny web app provided by the authors.
-
TARGET dataset of extracranial neuroblastoma (bulk RNA-seq) analyzed in this study were obtained as count matrices from the public Genomic Data Commons (GDC) cancer portal as described in R markdown
08.
GitHub / version control
The following are tracked / available on GitHub:
.Rmdfiles, containing the code, and.mdand rendered HTML files, containing code and outputs- The lockfile produced by the
renvpackage
The following are not tracked / available on GitHub:
- Figures in
png/pdfformat - Raw data and analysis output / processed data files (see Data availability section above)
- The actual packages in the R library
Citation
If you use or modify code provided here, please cite this work as follows:
Selin Jessa, Bhavyaa Chandarana, Steven Hébert, and Claudia L. Kleinman. (2024). NB-FOXR2 analysis code. Zenodo. https://www.doi.org/10.5281/zenodo.13755695