snps_mags Pipeline

snps_mags is a Nextflow pipeline designed for calling point mutations in Metagenome-Assembled Genomes (MAGs) using InStrain. The pipeline integrates state-of-the-art tools such as BWA-MEM2 for read alignment, SAMtools for BAM file processing, Qualimap for quality assessment, and MultiQC for comprehensive reporting. It is optimized for high-throughput analysis and can be executed in various environments using configurable execution profiles.

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Key Features

• Point Mutation Detection: Utilizes InStrain to identify single nucleotide variants (SNVs) in MAGs.
• End-to-End Workflow: From read alignment to variant calling and quality control.
• Containerized Execution: Leverages Singularity containers for reproducibility and portability.
• Scalable: Supports execution on SLURM clusters for large-scale analyses.
• Comprehensive Reporting: Generates detailed reports using MultiQC for easy interpretation of results.

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Overview

The pipeline performs the following tasks:

1. BWA Indexing: Indexes the reference genome using bwa-mem2.
2. SAMtools Indexing: Indexes the reference genome using samtools.
3. Alignment: Aligns paired-end reads to the reference genome using bwa-mem2, sorts the output, and marks duplicates.
4. Qualimap: Runs Qualimap to assess the quality of the aligned BAM files.
5. InStrain Variant Calling: Performs variant calling using InStrain.
6. MultiQC: Generates a MultiQC report summarizing the results.

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Pipeline Processes

1. bwa_index

• Description: Indexes the reference genome using bwa-mem2.
• Input: Reference genome file.
• Output: BWA index files .
• Container: oras://community.wave.seqera.io/library/bwa-mem2_instrain_multiqc_qualimap_samtools:850f96dbd001458f.

2. samtools_index

• Description: Indexes the reference genome using samtools.
• Input: Reference genome file.
• Output: SAMtools index file (.fai).
• Container: oras://community.wave.seqera.io/library/bwa-mem2_instrain_multiqc_qualimap_samtools:850f96dbd001458f.

3. aln_pipe

• Description: Aligns paired-end reads to the reference genome using bwa-mem2, sorts the output, and marks duplicates.
• Input:
  • Sample name and paired-end reads.
  • BWA index files.
• Output:
  • Marked BAM file (.marked.bam).
  • BAM index file (.marked.bam.bai).
  • Log file (.log.bwamem).
• Container: oras://community.wave.seqera.io/library/bwa-mem2_instrain_multiqc_qualimap_samtools:850f96dbd001458f.

4. qualimap

• Description: Runs Qualimap to assess the quality of the aligned BAM files.
• Input:
  • Sample name and marked BAM file.
  • Reference genome file.
• Output: Qualimap results directory.
• Container: oras://community.wave.seqera.io/library/bwa-mem2_instrain_multiqc_qualimap_samtools:850f96dbd001458f.

5. instrain_variant_calling

• Description: Performs variant calling using InStrain.
• Input:
  • Sample name and marked BAM file.
  • Reference genome file.
  • SAMtools index file (.fai).
• Output: InStrain results directory.
• Container: oras://community.wave.seqera.io/library/bwa-mem2_instrain_multiqc_qualimap_samtools:850f96dbd001458f.

6. multiqc

• Description: Generates a MultiQC report summarizing the results.
• Input: All output files from previous processes.
• Output: MultiQC report (.html).
• Container: oras://community.wave.seqera.io/library/bwa-mem2_instrain_multiqc_qualimap_samtools:850f96dbd001458f.

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Parameters

The pipeline is configured using the following parameters:

Parameter	Default Value	Description
reads	"data/*_{1,2}.fastq.gz"	Path to input paired-end reads.
reference	"reference.fa"	Path to the reference genome file (set of MAGs).
outdir	"results"	Directory to store pipeline outputs.
rlibrary	"pair-ends"	Read group library name.
rplat	"ILL"	Read group platform.
debug	true	Debug mode. If true, commands are printed but not executed.

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Resource Allocation

Resource allocation for specific processes can be configured as follows:

Process	Memory	CPUs
aln_pipe	5.GB	2
qualimap	5.GB	4
instrain_variant_calling	5.GB	4
multiqc	5.GB	1

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Execution Profiles

The pipeline supports multiple execution profiles for different environments:

1. kutral

• Executor: SLURM
• Queue: ngen-ko
• Queue Size: 10
• Container: Singularity
• Bind Path: /mnt/beegfs/labs/

2. leftraru

• Executor: SLURM
• Queue: slims
• Queue Size: 200
• Container: Singularity

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Execution Reports

The pipeline generates the following execution reports:

• Timeline: HTML file showing the execution timeline.
• Report: HTML file summarizing the execution.
• Trace: Text file containing execution trace information.
• DAG: HTML file showing the pipeline's Directed Acyclic Graph (DAG).

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Running the Pipeline

To run the pipeline, use the following command:

nextflow run pipeline.nf -profile <profile_name>

Replace <profile_name> with the desired execution profile (kutral or leftraru).

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Debug Mode

To run the pipeline in debug mode (commands are printed but not executed), set the debug parameter to true:

nextflow run pipeline.nf -profile <profile_name> --debug true

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Output Directory Structure

The pipeline outputs are organized as follows:

results/
├── bwa_index/
├── samtools_index/
├── alignment_results/
├── qualimap/
├── instrain/
├── multiqc/
└── pipeline_info/
    ├── execution_timeline_<timestamp>.html
    ├── execution_report_<timestamp>.html
    ├── execution_trace_<timestamp>.txt
    └── pipeline_dag_<timestamp>.html

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Notes

Ensure that the input files (reads and reference) are correctly specified in the params section.

Adjust resource allocation (memory and CPUs) based on your system's capabilities.

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Manifest

Key	Value
defaultBranch	main
homePage	https://github.com/digenoma-lab/snps_mags
author	Alex Di Genova
version	0.0.1