pgc-edc-oyster

GitHub Pages: Oyster scRNA‑seq Explorer

An interactive, static explorer is available in docs/ and can be published via GitHub Pages. It visualizes UMAP/t‑SNE embeddings from Cell Ranger analysis and colors cells by clusters.

Data wiring

The explorer automatically references these paths for each dataset:

• outs/analysis/umap/2_components/projection.csv
• outs/analysis/tsne/2_components/projection.csv
• outs/analysis/clustering/graphclust/clusters.csv

Datasets currently included:

• oyster_r1and2_CP3_roslin-mito-CRv3
• oyster_r1and2_CP2_roslin-mito-CRv3
• oyster_r1and2_CP1_roslin-mito-CRv3
• oyster_r1and2_Bla_roslin-mito-CRv3
• oyster_E4_redo2_roslin-mito
• oyster_E3_redo2_roslin-mito
• oyster_E2_redo2_roslin-mito
• oyster_E1_redo2_roslin-mito

Optional: Gene expression visualization

Expression layers are not bundled for page weight reasons. To add expression:

1. Export per‑cell expression for a set of genes into CSV files with headers Barcode,<GENE>.
2. Place them under docs/data/<dataset>/<gene>.csv and extend loadCoordinates/loadClusters with a loadExpression function.
3. Wire the Show button in docs/index.html to overlay expression values as a color scale.

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Repository associated with manuscript:

Primordial Germ Cell Specification and Early Developmental Cell States in Pacific Oyster

Mackenzie R. Gavery, Lauren Vandepas, Lauren M. Saunders, Brent Vadopalas, J. Adam Luckenbach, Cole Trapnell, Steven Roberts. Primordial germ cell specification and early developmental cell states in Pacific oyster. BMC Genomics 26, 951 (2025). https://doi.org/10.1186/s12864-025-12122-7

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Repository Structure

This repository contains single-cell RNA sequencing (scRNA-seq) analysis data and scripts for studying primordial germ cell specification and early developmental cell states in Pacific oyster (Crassostrea gigas).

Main Directory: oyster_scRNASeq_jobs_genomic_resources_outs/

1. annotation_files_script_forMonocle3/

Contains C. gigas gene annotation files used for Monocle3 analysis:

• Gene annotation tables with LOC identifiers
• Homology tables linking to other species (e.g., Strongylocentrotus purpuratus)
• R script for joining annotation files (annotations_joining_SRhomologtable.R)
• NCBI gene table downloads

Key files:

• LOCannot_homologies.txt - Gene homology annotations
• GCF_902806645.1_genetable_downloaded_011924.tsv - NCBI gene table
• Cg_hits-red_SpurSupp.tab - Cross-species gene comparisons

2. CellRanger_outputs_nobam/

Output files from 10x Genomics CellRanger analysis (BAM files excluded to save space):

• Gastrula stage samples: oyster_E1_redo2_roslin-mito through oyster_E4_redo2_roslin-mito
• Cleavage and blastula samples:
  • oyster_r1and2_Bla_roslin-mito-CRv3 (Blastula)
  • oyster_r1and2_CP1_roslin-mito-CRv3 through oyster_r1and2_CP3_roslin-mito-CRv3 (Cleavage period)

These directories contain filtered/raw feature-barcode matrices and analysis outputs used as input for Monocle3 analysis.

3. Monocle_Rscripts_RDS/

Monocle3 analysis scripts and RDS (R data serialization) files:

Main analysis scripts:

• monocle_gastrula_manuscript.R - Gastrula stage analysis
• monocle_CP_manuscript.R - Cleavage period analysis
• monocle_bla_manuscript.R - Blastula stage analysis
• monocle_CPbla_manuscript.R - Combined cleavage and blastula analysis
• monocle_figure_April2025.R - Figure generation script
• monocle_subcluster_analysis.R - Subcluster analysis
• MarkerGenes_markerscore.R - Marker gene scoring analysis
• scRNA_dotplot_clustered.R - Dot plot visualization
• barplot_semiquant.R - Semi-quantitative bar plots

RDS data objects:

• oyster_CP_only.RDS - Cleavage period cell dataset
• oyster_blastula_only.RDS - Blastula stage cell dataset
• oyster_cleavage_blastula.RDS - Combined dataset

Subdirectories:

• RDSgeneration_scripts_andRDSfiles/ - Scripts for generating RDS objects
• MarkerScoreAnalysis/ - Marker gene analysis results (top markers by score for different stages)

4. sc_gigas_jobs_genomicresources/

CellRanger job scripts and genome reference files:

Job scripts:

• count_CellRanger_E1_redo2.job through count_CellRanger_E4_redo2.job - Gastrula samples
• count_CellRanger_Bla_Roslin-mito.job - Blastula sample
• count_CellRanger_CP1_Roslin-mito.job through count_CellRanger_CP3_Roslin-mito.job - Cleavage period samples
• CellRanger_mkref_Roslin.job - Reference genome creation

Subdirectory:

• Cgigas_Roslin_mkref_genome-CRv3/ - CellRanger genome reference (STAR index and GTF files)

Note: Large genome/reference files (.fa, .gtf, STAR index files) are excluded via .gitignore

5. SRA_submission_docs/

Sequence Read Archive (SRA) submission metadata files:

• Sample metadata spreadsheets
• FASTQ file metadata and quality checks
• Multiple versions tracking submission revisions

Note: These files document data submission but are not required for reproducing the analysis.

6. manuscript_docs/

Supplementary materials for the manuscript:

• supplementary_figures_singlecell_oyster.docx - Supplementary figures
• supplementary_files_singlecell_oyster.xlsx - Supplementary data tables

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Additional Files

• pgc-edc-oyster.Rproj - RStudio project file
• .gitignore - Excludes large genome files, CellRanger outputs, and R workspace files

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Data Analysis Workflow

1. CellRanger processing: Raw sequencing data processed using scripts in sc_gigas_jobs_genomicresources/
2. Gene annotation: Annotation files prepared in annotation_files_script_forMonocle3/
3. Monocle3 analysis: Cell clustering, trajectory analysis, and marker identification using scripts in Monocle_Rscripts_RDS/
4. Visualization: Figures generated using various R scripts for manuscript preparation