PhyloPHoeNIx: Pipeline for relatedness determination using PHoeNIx output.

The latest version of PhyloPHoeNIx is currently in beta testing - please open an issue to report bugs

Metrics

Introduction

PhyloPHoeNIx is meant to be run in tandem with PHoeNIx to aid in outbreak investigations by estimating relatedness between samples. Both pipelines were built and are maintained by bioinformatians in the CDC's Division of Healthcare Quality Promotion (DHQP) to standardize surveillance of antibiotic resistance threats, identification of novel resistance threats and support public health laboratories in their genomic analysis of healthcare-associated infection organisms. PhyloPHoeNIx is a comprehensive pipeline that performs:

• Automation of DHQP's iterative outbreak analysis (runs analysis on all samples and when --by_st is passed also separates isolates by ST and runs each group through the analysis)
  
• SNV (single-nucleotide variant) matrix creation
  
• Phylogenetic tree building
  
• Reports the % core genome used in the SNV determination
  

cdcgov/phylophoenix is a bioinformatics best-practice analysis pipeline for relatedness determination using PHoeNIx output.

The pipeline is built using Nextflow, a workflow tool to run tasks across multiple compute infrastructures in a very portable manner. It uses containers making installation trivial and results highly reproducible. The Nextflow DSL2 implementation of this pipeline uses one container per process which makes it much easier to maintain and update software dependencies.

Quick Start

1. Install Nextflow (>=22.10.1)

2. Install any of Docker or Singularity (you can follow this tutorial) for full pipeline reproducibility.

3. Download the pipeline and test it on a minimal dataset with a single command:

bash nextflow run cdcgov/phylophoenix -profile test,singularity --outdir <OUTDIR> Note that some form of configuration will be needed so that Nextflow knows how to fetch the required software. This is usually done in the form of a config profile (YOURPROFILE in the example command above). You can chain multiple config profiles in a comma-separated string.

• The pipeline comes with config profiles called docker and singularity which instruct the pipeline to use the named tool for software management. For example, -profile test,docker.
• Please check nf-core/configs to see if a custom config file to run nf-core pipelines already exists for your Institute. If so, you can simply use -profile <institute> in your command. This will enable either docker or singularity and set the appropriate execution settings for your local compute environment.
• If you are using singularity, please use the nf-core download command to download images first, before running the pipeline. Setting the NXF_SINGULARITY_CACHEDIR or singularity.cacheDir Nextflow options enable you to store and re-use the images from a central location for future pipeline runs.

1. Start running your own analysis!

Pipeline overview

| PhyloPHoeNIx | SNVPhyl | | ------- | ------- | | | |

Running PhyloPHoeNIx

Inputs

You can either input samples using a typical nextflow samples sheet. This samplesheet should have the columns sample,directory. Here the directory would be to a sample's directory within a larger PHoeNIx output directory. For example, your samplesheet should look like this:

sample,directory 2023BB-00546,/PATH/PHX_output_dir/2023BB-00546 2023BB-00847,/PATH/PHX_output_dir/2023BB-00847

This method is useful for combining samples from different PHoeNIx directories and the full command would look like this:

bash nextflow run cdcgov/phylophoenix -profile singularity --outdir <OUTDIR> --input Directory_samplesheet.csv

Alternatively, if you want to analyze all samples in one PHoeNIx output directory you can pass the path to --input_dir and a samplesheet as described above will be created from all samples in this directory. Using this method the full command would look like this:

bash nextflow run cdcgov/phylophoenix -profile singularity --outdir <OUTDIR> --indir <PATH TO PHOENIX DIR>

By default PhyloPHoeNIx creates a SNV matrix and phylogenetic tree from all samples that pass QC in the directory. If you want to also group samples by MLST and create additional SNV matrices and phylogenetic trees for each MLST pass the --by_st argument.

bash nextflow run cdcgov/phylophoenix -profile singularity --outdir <OUTDIR> --input Directory_samplesheet.csv --by_st

If you want to skip running all samples together and just want to run in --by_st mode add the --no_all parameter.

bash nextflow run cdcgov/phylophoenix -profile singularity --outdir <OUTDIR> --input Directory_samplesheet.csv --by_st --no_all

A minimum of 3 samples of the same MLST are required to create a SNV matrix and phylogenetic tree.

Outputs

Here is an example output file tree that is reduced for space (3 samples are the min for creating a phylogenetic tree).

phylophoenixoutput
<ST>
<sampleid>
<sampleid>.bam
<sampleid>_sorted.bam
<sampleid>_filtered_density.txt
<sampleid>_freebayes_filtered.vcf.gz
<sampleid>_freebayes.vcf
<sampleid>_mpileup.vcf.gz
<ST>_centroid_info.txt
<ST>_cleaned_metadata.tsv --> upload to Microreact
<ST>_SNVPhyl.newick --> upload to Microreact/iTol or another visualisation program
<ST>_snvAlignment.phy
<ST>_snvMatrix.tsv --> upload to Microreact
<ST>_vcf2core.tsv
filtereddensityall.txt
filterStats.txt
mappingQuality.txt
newinvalidpositions.bed
TreeStatsSNVPhyl.txt
STSampleSheets
<ST>_samplesheet.csv
pipelineinfo
executionreport.html
executiontimeline.html
executiontrace.txt
pipelinedag.html
samplesheet.valid.csv
softwareversions.yml
Directorysamplesheet.csv
SNVPhylGRiPHinSummary.xlsx
GRiPHinSummary.xlsx

Credits

The core of PhyloPHoeNIx is a pipleline originally developed by @apetkau (Aaron Petkau), called SNVPhyl. There are standalone Nextflow, Galaxy, and [WDL] versions of SNVPhyl if you prefer to use those. For documentation on SNVPhyl see the following links.

• SNVPhyl Paper
• SNVPhyl Documenation

We thank the following people for their extensive assistance and test in the development of this pipeline:

• Nick Vlachos @nvlachos
• Thao Masters @masters-thao
• Alyssa Kent @Alyssa-Kent

Add beta testers here....

Contributions and Support

If you would like to contribute to this pipeline, please see the contributing guidelines.

For further information or help, don't hesitate to get in touch on the Slack #phylophoenix channel (you can join with this invite).

Citations

An extensive list of references for the tools used by the pipeline can be found in the CITATIONS.md file.

You can cite the nf-core publication as follows:

The nf-core framework for community-curated bioinformatics pipelines.

Philip Ewels, Alexander Peltzer, Sven Fillinger, Harshil Patel, Johannes Alneberg, Andreas Wilm, Maxime Ulysse Garcia, Paolo Di Tommaso & Sven Nahnsen.

Nat Biotechnol. 2020 Feb 13. doi: 10.1038/s41587-020-0439-x.

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