MobiCT is an analysis pipeline designed for detecting SNVs (Single Nucleotide Variants) and small InDels in circulating tumor DNA (ctDNA) obtained through non-invasive liquid biopsy. The pipeline serves diagnostic, prognostic, and therapeutic purposes in precision oncology.

The pipeline is built using Nextflow, a workflow tool to run tasks across multiple compute environments in a very portable manner. It uses Docker/Singularity containers making installation trivial and results highly reproducible.

MobiCT performs the following key steps:

1. Quality Control: Raw read quality assessment using FastP
2. Alignment: Read mapping to reference genome using BWA-MEM
3. Deduplication: PCR duplicate removal using Picard/fgbio
4. Variant Calling: SNV and InDel detection using VarDict
5. Annotation: Variant annotation using Ensembl VEP
6. Quality Metrics: Comprehensive QC metrics generation
7. Reporting: MultiQC report generation

1. Install Nextflow (>=20.04.0)

2. Create a conda environment with required tools:

   conda create -n mobict -c conda-forge -c bioconda \
     gatk4 fgbio bwa fastp samtools picard vardict ensembl-vep

3. Download the pipeline:

   git clone https://github.com/SimCab-CHU/MobiCT.git
   cd MobiCT

4. Test the pipeline with minimal dataset:

   conda activate mobict
   nextflow run MobiCT.nf -c nextflow.config --input test_data

nextflow -log /path/to/output/my.log run MobiCT.nf -c nextflow.config

Before running the pipeline, edit the nextflow.config file to specify:

• Input FASTQ files paths
• Output directory
• Reference genome path
• Target intervals/BED files
• Resource allocation

The pipeline expects:

• FASTQ files: Paired-end sequencing data from ctDNA samples
• Reference genome: Human reference genome (e.g., GRCh38)
• Target intervals: BED file defining regions of interest
• VEP database: Pre-downloaded VEP cache and databases

1. Download reference genome (GRCh38 recommended):

   wget http://ftp.ensembl.org/pub/release-104/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz

2. Download VEP databases (see VEP documentation):

   vep_install -a cf -s homo_sapiens -y GRCh38 -c /path/to/vep/cache

Results are organized in the specified output directory:

outdir/
├── sample1/
│   ├── sample1.deduplicated.bam
│   ├── sample1.deduplicated.bam.bai
│   ├── sample1.annotated.vcf
│   ├── sample1.HsMetrics.1.txt
│   ├── sample1.HsMetrics.3.txt
│   └── sample1.QC.bcftools_stats.stats
├── sample2/
│   └── ...
└── multiqc/
    └── multiqc_report.html

• .deduplicated.bam: Aligned, deduplicated BAM file
• .annotated.vcf: Variant calls with functional annotations
• .HsMetrics.*.txt: Hybrid selection metrics from Picard
• .QC.bcftools_stats.stats: Variant calling statistics
• multiqc_report.html: Comprehensive quality control report

The pipeline supports different execution profiles:

• conda: Use Conda for dependency management
• docker: Use Docker containers
• singularity: Use Singularity containers
• test: Run with test dataset

Example:

nextflow run MobiCT.nf -profile conda,test

Raw sequencing data (FASTQ files) of commercial controls used in the study are available at: NCBI SRA: PRJNA1209006

If you use MobiCT for your analysis, please cite:

MobiCT: ctDNA Analysis Pipeline

[Publication DOI will be added]

This pipeline uses several bioinformatics tools. Please also cite:

• Nextflow: Paolo Di Tommaso, et al. Nextflow enables reproducible computational workflows. Nature Biotechnology 35, 316–319 (2017)
• BWA: Li H. and Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754-1760 (2009)
• VarDict: Zhongwu Lai, et al. VarDict: a novel and versatile variant caller for next-generation sequencing in cancer research. Nucleic Acids Research 44, e108 (2016)
• VEP: McLaren W, et al. The Ensembl Variant Effect Predictor. Genome Biology 17, 122 (2016)
• MultiQC: Philip Ewels, et al. MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics 32, 3047-3048 (2016)

MobiCT was developed by the SimCab team at CHU.

This project is licensed under the MIT License - see the LICENSE file for details.

For questions and support:

• Create an issue on GitHub
• Contact the development team

• Initial release
• Support for SNV and small InDel detection in ctDNA
• Integrated quality control and reporting
• VEP-based variant annotation