Inputs and Data Preparation

Author: SeekGene

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Updated: 2026-10-13

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scMethyl + RNA-seq Inputs

Required Input Files

SeekSoul™ Methyl Tools accepts transcriptome and methylation paired-end FASTQ files for each sample:

• Transcriptome Read1 FASTQ
• Transcriptome Read2 FASTQ
• Methylation Read1 FASTQ
• Methylation Read2 FASTQ

If one sample contains multiple sequencing datasets, list them in the same order. In shell mode, separate file paths with commas. In Nextflow mode, provide one row per dataset in the samplesheet.

Shell Input Parameters

The shell wrapper sc_methy_workflow.sh uses the following arguments:

• $1: transcriptome Read1 FASTQ path
• $2: transcriptome Read2 FASTQ path
• $3: methylation Read1 FASTQ path
• $4: methylation Read2 FASTQ path
• --sample: sample name
• --outdir: output directory
• --database_dir: reference genome database directory
• --chemistry: DD-MET3 or DD-MET5 (default: DD-MET5)
• --core: number of CPU cores
• --filter_ch: remove reads with more than n CH methylation sites; set to 0 to disable

Sample Naming Rules

From v2.0.0 onward, sample_id and outdir are validated more strictly:

• Whitespace is not allowed
• Whitespace-like separators should be normalized to underscores
• Keep sample names stable across transcriptome and methylation inputs

Nextflow Samplesheet Format

The required header is:

sample_id,expression_r1,expression_r2,methylation_r1,methylation_r2

Example for one dataset:

sample_id,expression_r1,expression_r2,methylation_r1,methylation_r2
XYRD-WTJW880,/path/to/XYRD-WTJW880-E_S1_L005_R1_001.fastq.gz,/path/to/XYRD-WTJW880-E_S1_L005_R2_001.fastq.gz,/path/to/XYRD-WTJW880-MET_S01_L001_R1_001.fastq.gz,/path/to/XYRD-WTJW880-MET_S01_L001_R2_001.fastq.gz

Example when transcriptome and methylation FASTQ counts are not equal:

sample_id,expression_r1,expression_r2,methylation_r1,methylation_r2
WTJW969,/path/to/WTJW969_E_L003_R1.fq.gz,/path/to/WTJW969_E_L003_R2.fq.gz,/path/to/WTJW969_Met_L000_R1.fq.gz,/path/to/WTJW969_Met_L000_R2.fq.gz
WTJW969,/path/to/WTJW969_E_L004_R1.fq.gz,/path/to/WTJW969_E_L004_R2.fq.gz,/path/to/WTJW969_Met_L001_R1.fq.gz,/path/to/WTJW969_Met_L001_R2.fq.gz
WTJW969,,,/path/to/WTJW969_Met_L002_R1.fq.gz,/path/to/WTJW969_Met_L002_R2.fq.gz
WTJW969,,,/path/to/WTJW969_Met_L003_R1.fq.gz,/path/to/WTJW969_Met_L003_R2.fq.gz
WTJW969,,,/path/to/WTJW969_Met_L004_R1.fq.gz,/path/to/WTJW969_Met_L004_R2.fq.gz

Nextflow Run Parameters

Key parameters and profiles for running the Nextflow pipeline:

• --chemistry: methylation chemistry, DD-MET3 or DD-MET5 (default: DD-MET5)
• --expected_cell_num: expected number of cells used by the methy_only cell estimation (default: 3000)
• -profile docker: uses the public container image ghcr.io/seekgene/seeksoulmethyl_docker:v1.0.0
• -profile aliyun_k8s and -profile aliyun_k8s_argo: use the internal Aliyun registry image seekgene-registry-vpc.cn-beijing.cr.aliyuncs.com/seekgene/seeksoulmethyl:1.1.2

Reference Database Requirements

The --database_dir directory should contain:

<database_dir>/
|-- bed/
|   |-- chr_len.bed
|   `-- chr_nochrM.bed
|-- fasta/
|   |-- genome.fa
|   |-- genome.fa.fai
|   `-- Bisulfite_Genome/
|-- genes/
|   `-- genes.gtf
`-- star/

Path conventions used by the pipeline:

• star/: STAR genome index
• fasta/genome.fa: genome FASTA
• fasta/: Bismark genome folder after bismark_genome_preparation
• genes/genes.gtf: gene annotation
• bed/chr_len.bed: chromosome sizes
• bed/chr_nochrM.bed: recommended chromosome list without mitochondrial contigs

For detailed construction steps, see:

• Reference genome database guide
• How to build the reference genome database

Repository Layout

After cloning, the key Nextflow entry points and modules are:

• nf/main.nf: Top-level entry. Select sub-workflow via --workflow (rna_met, methy_only, force_cell).
• nf/subworkflows/: Workflow definitions:
  • rna_met/main.nf: Transcriptome + methylation end-to-end processing.
  • met_only/main.nf: Methylation-only workflow.
  • forcecell/main.nf: Force-cell workflow (recomputes/updates results using previous outputs).
• nf/modules/: Step-wise process modules:
  • step1.nf: preprocessing, QC, barcode extraction, transcriptome analysis.
  • step2.nf: Bismark alignment and BAM sorting.
  • step3.nf: per-cell BAM splitting, ALLC generation/merge, multi-scale datasets.
  • step4.nf: summaries, dimensionality reduction, joint report.
  • utils.nf: helpers for methylation-only workflow (reads counting and cell estimation).
• nf/bin/: Helper scripts and resources (e.g., barcode whitelists).
• nf/nextflow.config: Executors and resource configuration.
• nf/nextflow_schema.json: Pipeline parameter schema.
• sc_methy_workflow.sh: Shell script to run the dual-omics analysis pipeline.