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 the data/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 analysis
• code --> code for R analysis, contains the .Rmd files that run the high-level analyses and produce figures included in the paper
  • functions --> contains .R files with custom functions used throughout the analysis
  • scripts --> 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 repository
• rr_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:

• .Rmd files, containing the code, and .md and rendered HTML files, containing code and outputs
• The lockfile produced by the renv package

The following are not tracked / available on GitHub:

• Figures in png/pdf format
• 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