Spectronaut-nf

A nextflow pipeline to carry Spectronaut DIA analysis with its parallel execution attribute. Here, individual LC-MS/MS derived proteomic rawfiles are queued for Spectronaut search separately with the help of nextflow parallel execution attribute and the results are combined in the end to form an experiment wide output. This increases the Spectronaut DIA analysis search speed owing to the use of multiple High-Performance Computing (HPC) nodes with the help of batch schedulers (ex: SLURM).

Key Features

• 🚀 Parallel execution of Spectronaut searches across multiple HPC nodes
• 📈 Scalable processing of hundreds to thousands of DIA raw files
• ⚡ Reduced analysis time compared with standard Spectronaut execution
• 🔄 Fully resumable workflow using Nextflow caching
• 🖥️ Compatible with HPC schedulers such as SLURM
• 🧩 Configurable computational resources for each workflow stage
• 📊 Generates experiment-wide spectral libraries and DIA reports

Spectronaut-nf workflow

The pipeline consists of six main processes:

1. Library generation - Pulsar Stage 1
2. Generate QSP
3. Library generation - Pulsar Stage 3
4. Combine PSAR
5. DIA search
6. Combine/merge SNE

Installation

clone Spectronaut-nf repository

git clone https://github.com/BenCollinsLab/Spectronaut-nf
cd Spectronaut-nf

Prerequisite

The following software packages must be installed and accessible in your environment before running Spectronaut-nf.

Software	Minimum Version	Purpose
.NET	8.0.18	Required to execute the Spectronaut command-line application (SpectronautCMD.dll).
Nextflow	25.10.3	Workflow manager used to orchestrate and execute the pipeline processes.
SLURM	24.04.3	Optional but recommended HPC workload manager used to distribute and execute jobs in parallel across compute nodes.

Verify Installation

$ dotnet --info

Host:
  Version:      8.0.18
  Architecture: x64
  Commit:       ef853a7105
  RID:          rocky.8-x64
.
.
.

$ nextflow info
  Version: 25.10.3 build 10983
  Created: 22-01-2026 15:34 UTC (15:34 BST)
  System: Linux 4.18.0-553.64.1.el8_10.x86_64
  Runtime: Groovy 4.0.28 on Java HotSpot(TM) 64-Bit Server VM 22.0.2+9-70
  Encoding: UTF-8 (UTF-8)

$ sinfo --version
slurm 25.05.2

Note: Spectronaut-nf has been developed and tested using the software versions listed above. Earlier or later versions may work but have not been extensively validated.

Cluster Environment: On most HPC systems, these dependencies can be loaded through environment modules, for example:

module load dotnet/8.0.18
module load nextflow/25.10.3
module load slurm/25.05.2

Please consult your local HPC administrator if different module names or versions are used on your system.

Spectronaut-nf input variables

Users can set most of the input parameters required to run the Spectronaut-nf pipeline in params.yaml file. This file can be edited using vim or nano editor in linux/HPC platform.

DIA search inputs

Required Parameters

Define or modify all DIA search-related inputs in params.yaml:

Parameter	Description
JOB_NAME	Unique identifier/name for your Spectronaut search job
spec_bin	Absolute path to the Spectronaut command-line executable (SpectronautCMD.dll)
license	Absolute path to your Spectronaut license file
rawfile_dir	Directory path containing all input LC-MS/MS raw data files
FASTA	Protein sequence database file in bgsfasta format for peptide identification
EXT_PSAR	External Spectronaut result file (.psar) to use as input for DIA search

Optional Parameters

Parameter	Description
sample_size	Number of raw files to randomly sample for library generation (if not specified, all files are used)
batch_size	Number of raw files to group together for each library generation process (i.e., task). Default: 1 (individual files processed separately via Pulsar)
excludePattern	Exclude raw files by name pattern (e.g., "pooled" will skip files containing "pooled")
COND_SETUP	TSV file specifying experimental condition/sample grouping metadata
REPORT	Spectronaut report template (.rs file) to customize output report columns and format
PROP_DIA	Optional custom Spectronaut search parameters file (.prop) for advanced search configuration
max_files_for_ManageSNE	Maximum number of SNE result files threshold for manage SNE process execution. If the total number of raw files exceeds this cutoff, the pipeline will execute combineSNE process instead. Default: 150 (adjust based on available HPC/Cloud resources for large-scale SNE file merging)

HPC Configuration

Set the batch scheduler used in your HPC platform inside process.

executor = 'slurm'

This will be followed by setting up process-specific partition requirements such as CPUs, RAM and Duration available in the respective HPC partition.

Parameter	Description
*_queue	HPC queue/partition used for each task.
*_cpus	Number of CPU cores allocated per jtaskob.
*_memory	Memory allocated per task.
*_time	Maximum wall-clock time for task execution.

Note: Each process is executed by different modules in Spectronaut-nf i.e., SN_nf_pulsar (Process 1-3) , SN_nf_combine_psar (Process 4), SN_nf_dia (Process 5) and SN_nf_combine_sne (Process 6), each with configurable HPC resource requirements. Adjust the CPU, memory, and time limits based on your HPC cluster capabilities and data size.

Parallel Execution Control

• executor_queueSize - Maximum number of search tasks to execute in parallel on the HPC queue

Running Spectronaut-nf

nextflow -bg run /path/to/main.nf \
         -params-file /path/to/params.yaml >> nextflow_cmd.log

Resume Failed Runs

nextflow -bg run /path/to/main.nf \
         -params-file /path/to/params.yaml \
         -resume >> nextflow_cmd.log

Nextflow automatically reuses previously completed tasks and continues from the last successful checkpoint.

Output

Output Folder	Description
intermediates/	Task-specific outputs generated during the library creation stages (Pulsar Stage 1, Generate QSP, and Pulsar Stage 3).
out_lib/	Final library generation outputs, including the experiment-wide spectral library files (.psar and .kit) obtained by combining all task-specific .psar files.
out_dia/	Outputs from all DIA search tasks, including the final experiment-wide DIA search results. Users can use the final Spectronaut reports or SNE file for further downstream analysis.
logs/	Nextflow execution reports, timelines, traces, and other log files generated during the pipeline run.
work/	Nextflow working directory containing task-specific execution folders identified by unique hash keys.
tmp/	Temporary files and intermediate data generated during pipeline execution. These files can be safely removed after successful completion of the pipeline.

Example Directory Structure

results/
├── intermediates/
├── out_lib/
├── out_dia/
├── logs/
├── tmp/
└── work/

Citation

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