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Preparing Data & Samples

To run the pipeline on your own data, you need to organize your raw files and create a Sample Sheet CSV. This file maps your raw sequencing files to sample names and biological conditions.

1. Organizing Your Raw Files

The pipeline can work with FASTQ files anywhere on your system, but we recommend placing them in a data/ or reads/ directory within your project root.

  • Fastq files should be gzipped (.fastq.gz or .fq.gz).
  • Paired-end files typically use _R1 / _R2 or _1 / _2 suffixes.

2. Creating a Sample Sheet

Each sequencing library requires a corresponding CSV sample sheet. This file is the primary way the pipeline discovers your data.

Column Reference

The following columns are grounded in the pipeline's internal logic:

Column Required Description
name Yes A unique biological identifier for the sample (e.g., WT_Rep1).
filename SE The path to the raw sequence file for Single-End reads.
filename_1 PE The path to the first mate for Paired-End reads.
filename_2 PE The path to the second mate for Paired-End reads.
condition No* Metadata for DESeq2 (e.g., WT, KO). You can add any number of additional metadata columns.

Note

*While additional columns like condition are not strictly required for alignment, they are essential for downstream Differential Expression (DE) analysis.

Examples

Single-End (SE) Sample Sheet

name,filename,condition
Control_Rep1,reads/ctrl1.fq.gz,control
Control_Rep2,reads/ctrl2.fq.gz,control
Treated_Rep1,reads/treat1.fq.gz,treated

Paired-End (PE) Sample Sheet

name,filename_1,filename_2,treatment
Sample_A,data/sA_R1.fastq.gz,data/sA_R2.fastq.gz,Basal
Sample_B,data/sB_R1.fastq.gz,data/sB_R2.fastq.gz,Basal
Sample_C,data/sC_R1.fastq.gz,data/sC_R2.fastq.gz,TGFb

3. Library Preparation Reference

In your configuration, you will link these sample sheets to a library name and specify the protocol (se or pe).

sequencing_libraries:
  - name: "MyExperiment"
    protocol: "pe" # Must match your sample sheet columns!
    sample_sheet: "my_samples.csv"

Warning

If you specify protocol: "pe", the pipeline expects filename_1 and filename_2 in the sample sheet. If you specify protocol: "se", it expects filename.

Next Steps

Once your data is prepared, learn how to use Profiles to encapsulate all your run settings: