Sample Sheets in `slide_snake`

Sample sheets are the central configuration files that tell slide_snake how to process your spatial RNA-seq data. They provide flexibility in specifying different analysis parameters, reference genomes, and processing recipes for each sample.

Overview

The sample sheet is a CSV file that contains all the necessary information about your samples, including:

Input FASTQ file paths
Reference genome locations
Processing recipes to apply
Platform-specific parameters
Quality control settings

Quick Start

Use the template: Start with docs/example_sample_sheet.csv
Edit for your data: Modify paths and parameters for your samples
Update config: Set SAMPLE_SHEET_PATH in config.yaml to point to your sample sheet
Validate: Run snakemake -n to check for errors

Sample Sheet Columns

Required Columns

Column	Description	Example
`sampleID`	Unique identifier for each sample	`sample_001_visium`
`fastq_R1`	Path to Read 1 FASTQ file(s)	`data/sample1_R1.fq.gz`
`fastq_R2`	Path to Read 2 FASTQ file(s)	`data/sample1_R2.fq.gz`
`recipe`	Processing recipe(s) for short reads	`visium` or `visium seeker`
`species`	Species name for reference selection	`human`, `mouse`, `rat`

Platform Detection Columns

Column	Description	Example
`whitelist`	Path to barcode whitelist file	`resources/visium_whitelist/whitelist.txt`
`recipe_ONT`	Recipe(s) for Oxford Nanopore data	`visium_ont`

Reference Genome Columns

Column	Description	Required For	Example
`STAR_ref`	STAR genome index directory	STAR alignment	`/path/to/STAR_index/`
`kb_idx`	Kallisto index file	Kallisto alignment	`/path/to/transcriptome.idx`
`kb_t2g`	Transcript-to-gene mapping	Kallisto alignment	`/path/to/t2g.txt`
`genome_fa`	Reference genome FASTA	Various analyses	`/path/to/genome.fa`
`genes_gtf`	Gene annotation GTF file	Gene quantification	`/path/to/genes.gtf`
`cDNA_fa`	Transcript sequences FASTA	ONT transcriptome alignment	`/path/to/transcripts.fa`

Quality Control Columns

Column	Description	Example
`rRNA_ref`	rRNA reference for filtering	`/path/to/rRNA_ref.fa`
`rRNA_gtf`	rRNA annotation GTF file	`/path/to/rRNA.gtf`

File Path Specifications

Multiple Files

Specify multiple FASTQ files in several ways:

# Space-separated files
fastq_R1,fastq_R2
"file1_R1.fq.gz file2_R1.fq.gz","file1_R2.fq.gz file2_R2.fq.gz"

# Glob patterns (use with caution)
fastq_R1,fastq_R2
"data/sample*_R1.fq.gz","data/sample*_R2.fq.gz"

Path Types

Absolute paths: /full/path/to/file.fq.gz
Relative paths: data/file.fq.gz (relative to workflow directory)
Compressed files: .gz, .bz2 files are automatically handled

Recipe Specifications

Single Recipe

recipe
visium

Multiple Recipes

Use spaces to separate multiple recipes for benchmarking:

recipe
"visium visium_total"

This will run both standard and total RNA alignment strategies.

Species and Reference Configuration

Common species configurations:

Human (GRCh38)

species,STAR_ref,genes_gtf,kb_idx,kb_t2g
human,/path/to/GRCh38_STAR/,/path/to/gencode.v47.gtf,/path/to/human.idx,/path/to/human_t2g.txt

Mouse (GRCm39)

species,STAR_ref,genes_gtf,kb_idx,kb_t2g  
mouse,/path/to/GRCm39_STAR/,/path/to/gencode.vM36.gtf,/path/to/mouse.idx,/path/to/mouse_t2g.txt

Platform-Specific Examples

10x Visium

sampleID,fastq_R1,fastq_R2,recipe,whitelist,species,STAR_ref,genes_gtf,kb_idx,kb_t2g
visium_sample,data/visium_R1.fq.gz,data/visium_R2.fq.gz,visium,resources/visium_whitelist/whitelist.txt,human,/ref/GRCh38_STAR,/ref/gencode.v47.gtf,/ref/human.idx,/ref/human_t2g.txt

Curio Seeker

sampleID,fastq_R1,fastq_R2,recipe,whitelist,species,STAR_ref,genes_gtf,kb_idx,kb_t2g
seeker_sample,data/seeker_R1.fq.gz,data/seeker_R2.fq.gz,seeker_MatchLinker,data/barcodes.txt,mouse,/ref/GRCm39_STAR,/ref/gencode.vM36.gtf,/ref/mouse.idx,/ref/mouse_t2g.txt

DBIT-seq

sampleID,fastq_R1,fastq_R2,recipe,whitelist,species,STAR_ref,genes_gtf,kb_idx,kb_t2g
dbit_sample,data/dbit_R1.fq.gz,data/dbit_R2.fq.gz,dbit-pretrim,resources/dbit_whitelist/Spatial_barcode_100x100.txt,human,/ref/GRCh38_STAR,/ref/gencode.v47.gtf,/ref/human.idx,/ref/human_t2g.txt

Oxford Nanopore (ONT)

sampleID,fastq_R1,fastq_R2,recipe,recipe_ONT,whitelist,species,cDNA_fa,genes_gtf
ont_sample,data/ont_reads.fq.gz,,visium,visium_ont,resources/visium_whitelist/whitelist.txt,human,/ref/transcripts.fa,/ref/gencode.v47.gtf

Advanced Configuration

Custom Recipes

Create custom recipes by modifying resources/recipe_sheet.csv. See Recipes Documentation for details.

rRNA Filtering

Enable ribosomal RNA filtering:

rRNA_ref,rRNA_gtf
/path/to/rRNA_BWA_index,/path/to/rRNA_annotations.gtf

Quality Control Parameters

The pipeline automatically handles:

FastQC analysis on raw and processed reads
Alignment quality assessment with Qualimap
Read mapping statistics
Barcode correction statistics

Validation and Troubleshooting

Check Sample Sheet Format

# Dry run to validate sample sheet
snakemake -n --use-conda

# Check specific sample
snakemake -n out/{SAMPLE_ID}/illumina/STAR/{RECIPE}/counts_filtered/

Common Issues

Issue: File paths not found

Solution: Use absolute paths or verify relative paths from workflow directory

Issue: Recipe not recognized

Solution: Check recipe name exists in resources/recipe_sheet.csv

Issue: Reference files missing

Solution: Verify all reference paths exist and are readable

Issue: Multiple files not processed

Solution: Ensure proper quoting of space-separated file lists

Best Practices

Use absolute paths when possible to avoid path resolution issues
Test with small datasets before running full analyses
Keep consistent naming across samples for easier downstream analysis
Document your recipes if creating custom analysis parameters
Validate references ensure all reference files exist and are properly formatted
Use version control to track changes to your sample sheets

Example Sample Sheets

Complete Example

sampleID,fastq_R1,fastq_R2,recipe,recipe_ONT,whitelist,species,STAR_ref,genes_gtf,kb_idx,kb_t2g,rRNA_ref,rRNA_gtf,genome_fa,cDNA_fa
test_visium,data/visium_R1.fq.gz,data/visium_R2.fq.gz,visium,,resources/visium_whitelist/whitelist.txt,human,/ref/GRCh38_STAR,/ref/gencode.v47.gtf,/ref/human.idx,/ref/human_t2g.txt,/ref/rRNA_BWA,/ref/rRNA.gtf,/ref/GRCh38.fa,/ref/transcripts.fa
test_seeker,data/seeker_R1.fq.gz,data/seeker_R2.fq.gz,"seeker_MatchLinker seeker_std",,data/seeker_barcodes.txt,mouse,/ref/GRCm39_STAR,/ref/gencode.vM36.gtf,/ref/mouse.idx,/ref/mouse_t2g.txt,/ref/rRNA_BWA,/ref/rRNA.gtf,/ref/GRCm39.fa,/ref/mouse_transcripts.fa

This comprehensive sample sheet demonstrates multi-recipe analysis and complete reference specification.

Sample Sheets in slide_snake