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16S Guild Long

16Sguild: Guild-based 16S-rRNA sequencing analysis pipeline

A Nextflow workflow for guild-based 16S-rRNA sequencing analysis such as introduced by Wu and Zhao et al., 2021.

Workflow

Basic workflow overview of 16Sguild pipeline.

Quick Start

Run directly from GitHub (suggested):

nextflow run zhao-microbiome-lab/16Sguild/main.nf -params-file examples/params.yml

Or, clone the repository and launch the pipeline:

git clone https://github.com/zhao-microbiome-lab/16Sguild.git
cd 16Sguild
nextflow run main.nf -params-file examples/params.yml

Detailed usage examples can be found in the Basic Workflow documentation.

Note: The pipeline requires Nextflow (version 24.04.0 or later) and either Docker or Singularity/Apptainer.

Parameter Configuration

Parameters are supplied in a config file, e.g. params.yml. Here is an example of a completed params.yml file. At the end of each part, users will need to examine their data and fill in the required values before continuing with their analysis.

# Required initial parameters
samplesheet: "samplesheet.csv"
metadata: "metadata.tsv"
base_name: "base_name_dataset"
input_type: "SampleData[PairedEndSequencesWithQuality]"
input_format: "PairedEndFastqManifestPhred33"
trim_forward: "GTGCCAGCMGCCGCGGTAA"
trim_reverse: "GGACTACHVGGGTWTCTAAT"

# Required parameters updated based on data
# End of Part 1
trunc_forward: "xxx"
trunc_reverse: "xxx"
max: "xxx"

# End of Part 2
linkage_table: /path/to/file.txt

# End of Part 3
sampling_depth: "xxx"

An example parameters file is available in examples/params.yml.

Brief Parameter Descriptions

Parameter Description
samplesheet Path to the CSV file containing sample information
base_name String identifying the name of input folder
input_type Semantic type required for the QIIME2 import function
input_format Format of the data to import (e.g., "PairedEndFastqManifestPhred33")
trim_forward Forward primer sequence to trim
trim_reverse Reverse primer sequence to trim
metadata Path to the sample metadata table (TSV format)
trunc_forward Position at which to truncate forward reads in QIIME2 DADA2 (experiment-dependent)
trunc_reverse Position at which to truncate reverse reads in QIIME2 DADA2 (experiment-dependent)
linkage_table Table from 16Sguild database assigning UUIDs to ASVs (experiment-dependent)
max Maximum depth value used for alpha rarefaction (experiment-dependent)
sampling_depth Rarefaction sampling depth for diversity analysis (experiment-dependent)

Project Structure

  • main.nf – Main Nextflow pipeline
  • processes/ – Modular pipeline processes
  • 16SguildR/ – Custom R package for downstream analysis
  • bin/ – Helper scripts (ensure they are executable)
  • lib/ – Definition script for end of part output
  • docs/ – Documentation for 16Sguild pipeline
  • conf/ – Config profiles (e.g., for HPC, Docker, Singularity)
  • examples/ – Example input and config files
  • docker/ – Definition files for Singularity images automatically used in pipeline

Containers

This pipeline uses the following containers by default (defined in conf/default.config):

  • FastQC: docker://biocontainers/fastqc:v0.11.9_cv8
  • MultiQC: docker://multiqc/multiqc:latest
  • QIIME2: docker://zhao-microbiome-lab/16sguild:qiime2_2024.2_1.0.0
  • FastSpar: quay.io/biocontainers/fastspar:1.0.0--h7f8d780_0
  • R: docker://zhao-microbiome-lab/16sguild:r_env_1.0.9
  • Picrust2: quay.io/biocontainers/picrust2:2.6.2--pyhdfd78af_1

No manual installation of software is needed—these containers are pulled automatically.

Output

  • All results are written to the results/ directory by default.
  • results/main_results contains output files from the pipeline.
  • results/database contains the .rds file of the linkage table that can be uploaded to the database.
  • results/visualizations contains files that can be used on the QIIME2 viewing platform.
  • Intermediate files are preserved in the Nextflow work directory for reproducibility and in results/intermediate_results for easy access.
  • Results files are symlinked by default. Users can set publish.mode = 'copy' to change this.

Testing

Test the pipeline with example data:

nextflow run main.nf -profile test

The test profile runs a minimal dataset and can be used for continuous integration.

Support

  • For questions or issues, please open an issue.
  • For feature requests, submit a new issue with the enhancement label.

Citation

If you use this workflow, please cite: - This repository: zhao-microbiome-lab/16Sguild - Guild analysis: Zhao, Wu, & Zhao, 2024 - Nextflow: Di Tommaso et al. 2017

License

Distributed under the MIT License. See LICENSE for details.