About

TaxTriage a flexible, containerized bioinformatics pipeline for identification of pathogens within short- or long-read metagenomic sequence data, generated from complex samples/specimens (e.g., respiratory swabs, lesion swabs, whole blood). The workflow includes various software packages to perform quality control, classification, read mapping, as well as generation of confidence metrics and a final report listing anticipated pathogens of relevance.

Description

Tax Triage is designed as a pipeline to democratize metagenomic sequence analysis for use in public health for early warning, outbreak investigation, and potentially in clinical settings. To this end, TaxTriage was developed to ingest short- or long-read metagenomic sequencing data generated from tissues (human or animal). The intent is to provide non-bioinformaticians a tool capable of generating species-level identifications of pathogens from raw metagenomic or targeted sequencing data. Specific modules to be used depend on sequencing chemistry and settings are anticipated to vary based on specimen type. Strain, variant, or clade-level distinction may be possible with specialized datasets, but it is anticipated that level of granularity would require subsequent, specialized analyses.

• Quality control steps
• In-silico host depletion
• Classification of reads
• Mapping of reads to reference genomes found to be "top hits"
• Confidence metric generation (e.g., depth/breadth of coverage, %nt ID)
• Threshold mechanisms
• De-novo assembly
• Detailed MultiQC reports
• Concise final report (intended to have all data fields required for use in clinical settings)

For the purpose of giving an initial triage of taxonomic classifications, using Kraken2 database(s), that can then be ingested into a CLIA-style report format. It is under active development, but in the current state it is capable of running a set number of samples end-to-end using a user-created samplesheet in .csv format. The output format is a HTML which is highly interactive and distributable. This pipeline uses the nextflow ecosystem and is also available as a module in Basestack.

Currently, Basestack is undergoing improvements to allow easier usage of nextflow pipelines (includes TaxTriage) that is scheduled for release in early August.

See Important output locations for information on where to get the most important output files from the pipeline.

See here for information on how "top hits" is located

Alerts

⚠️ If you make changes to the code within a nextflow-pulled repo, a change can result in a conflict in updating already cloned repos when running the test profile or called -latest -r main/stable. As a result you must run nextflow drop https://github.com/jhuapl-bio/taxtriage first. This only applies to pipelines run by calling the remote repo and the previously mentioned parameters. If you expect to make local changes frequently, you should just git clone and git pull manually and run the pipeline from the main.nf file. See here for more info

The pipeline is built using Nextflow, a workflow tool to run tasks across multiple compute infrastructures in a very portable manner. It uses Docker/Singularity 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. Where possible, these processes have been submitted to and installed from nf-core/modules in order to make them available to all nf-core pipelines, and to everyone within the Nextflow community!

On release, automated continuous integration tests run the pipeline on a full-sized dataset on the AWS cloud infrastructure. This ensures that the pipeline runs on AWS, has sensible resource allocation defaults set to run on real-world datasets, and permits the persistent storage of results to benchmark between pipeline releases and other analysis sources. The results obtained from the full-sized test can be viewed on the nf-core website.

Tax Triage is designed as a pipeline for the purpose of giving an initial triage of taxonomic classifications, using Kraken2 database(s), that can then be ingested into a CLIA-style report format. It is under active development, but in the current state it is capable of running a set number of samples end-to-end using a user-created samplesheet in .csv format. The output format is a HTML which is highly interactive and distributable. This pipeline uses the nextflow ecosystem and is also available as a module in Basestack. Currently, Basestack is undergoing improvements to allow easier usage of nextflow pipelines (includes TaxTriage) that is scheduled for release in early August.

Efforts are underway to provide full support of this pipeline on nf-core to provide a seamless deployment methodology. The pipeline also requires installation of Docker or Singularity (CE ONLY v4+) for the individual modules within it. Because these modules are separate from the source code of TaxTriage, we recommend following the examples outlined in the usage details first to automatically run the pipeline and install all dependencies while also giving you some example outputs and a better feel for how the pipeline operates.

See Here for full usage details

See Here for troubleshooting & FAQ

Installation

TaxTriage requires 2 primary installs for it to work

1. Nextflow
2. Singularity or Docker (recommended)

1. Nextflow

Follow instructions here or run these commands in your WSL2, Native Linux, or Mac environment

# Make sure that Java v11+ is installed:
java -version
 
# Install Nextflow
curl -fsSL get.nextflow.io | bash

Note, this command requires sudo to move to your home path. If you are on an HPC, make sure that nextflow is in your $PATH if not globally available Place it in your $PATH

# Add Nextflow binary to your user's PATH:
mv nextflow ~/bin/

If installing globally, requiring sudo, type:

sudo mv nextflow /usr/local/bin

When complete, verify installation with nextflow -v to see the version

2. Containerization Approach Install

Choose A (Recommended - Docker) or B. If on a HPC, talk with your IT to get B. Singularity setup. You do NOT need to install both software tools.

A. Docker

Follow these steps for your OS here - IF on WSL2 (Windows), choose Docker Desktop for Windows and it should be available automatically in your WSL environment

B. Singularity

Install Instructions

Quick Start

Make sure you have either Docker or Singularity installed, as well as Nextflow

Test Run

This will pull the test data and run the pipeline. It should take ~10-15 minutes.

nextflow run https://github.com/jhuapl-bio/taxtriage -r main -latest -profile test,docker -resume

❗If you want singularity instead, make sure to specify that in the profile instead of docker like: test,singularity

Cloud

Follow the steps here

Offline Local Mode

In some cases, you may not want to always pull the latest update(s) each time your run the pipeline. To solve this issue, you have 2 primary options:

A. Reference remote url, don't specify latest

nextflow run https://github.com/jhuapl-bio/taxtriage -r main -profile test,docker -resume

Here, we remove the -latest which will not attempt to pull updates. This will only work if you've already run the pipeline (thus pulling the code locally) in online mode like in the initial example for a test run

B. Clone the repo first, reference local main file

Here, we instead clone the repo. Then, we reference the launchfile called main.nf that is locally on our system. We need to ensure that we're always in the repo's directory each time we do this too

First we clone

git clone https://github.com/jhuapl-bio/taxtriage.git

Then we cd into our directory

cd taxtriage

Finally, we run a test run (feel free to edit inputs based on your own data needs after the first test run)

nextflow run ./main.nf -profile test,docker -resume

Please be aware that intermittent portions of the pipeline will still use internet by default. You can instead run other commands like the example here to remedy this problem.

Local Data

⚠️ If you get an error on uncommitted changes please run the nextflow drop -f https://github.com/jhuapl-bio/taxtriage

nextflow drop -f https://github.com/jhuapl-bio/taxtriage

Then run the pipeline normally as described in previous steps

Running it with the local config (for laptops/workstations) with limited RAM and a different (auto-downloadable) db

nextflow run https://github.com/jhuapl-bio/taxtriage  \
  --outdir tmp_viral \
  -resume \
  --input examples/Samplesheet.csv \
  -r main -latest \
  --db "viral" --download-db \
  -profile local,docker

Running it by overriding some parameters from the local config

nextflow run https://github.com/jhuapl-bio/taxtriage \
   --input examples/Samplesheet.csv -r main -latest \
   --db viral --download_db \
   --outdir output_viral --max_memory 10GB --max_cpus 3   \
   -profile docker  -resume --remove_taxids "9606"

⚠️ Please see the contents of test or local config to figure out what the defaults are for those profiles

Remember, if you are doing a single taxid, wrap it with '' inside the "" quote

Using a db that is on your local filesystem

Make sure you use a local k2 database in your system. Assuming (for this example) you've pulled and decompressed a k2 database like k2_viral See here for more and change it with the --db parameter like below.

nextflow run https://github.com/jhuapl-bio/taxtriage \
   --input examples/Samplesheet.csv \
   --db "./k2_viral" -r main -latest \
   --outdir output_viral_local  \
   --profile local,docker \
   -resume

Note that the --db parameter is changed to a local path which contains the k2d files for kraken2 to operate.

Running it without internet availability

This will use a local assembly text and reference fasta, assuming the reference FASTA is called refer.fasta

You will need 3 files locally on your system

1. assembly
2. reference_fasta
3. db

nextflow run https://github.com/jhuapl-bio/taxtriage \
   --input examples/Samplesheet.csv \
   --db "k2_viral" -r main -latest --skip_kraken2 \
   --outdir tmp --reference_fasta ./refer.fasta \
   -profile local,docker \
   -resume \
   --demux \
   --assembly examples/assembly_summary_refseq.txt

Be aware that this skips the metagenomics portion of the pipeline and only does alignment using the local reference fasta.

Running on local nf files (test config)

⚠️ Make sure you're in the jhuaplbio/taxtriage repo first!

nextflow run ./main.nf -profile test,docker

See here for a full list of input parameters and options available based on your own needs

If you want to download the databases from scratch, you can see them here Make sure to Download these databases to your Desktop or wherever you are the most comfortable. Remember the location and specify the --db parameter as the absolute path. For example ~/Desktop/flukraken2. Also, remove the --download-db parameter

• standard-8
• viral
• flukraken2

Pipeline summary

1. Read QC (FastQC)
2. Present QC for raw reads (MultiQC)

Quick Start Highlight

1. Install Nextflow (>=21.10.3)

2. Install any of Docker, Singularity (you can follow this tutorial).

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

   nextflow run https://github.com/jhuapl-bio/taxtriage -profile test,docker --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 or 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 enables you to store and re-use the images from a central location for future pipeline runs.

4. Start running your own analysis!

           nextflow run https://github.com/jhuapl-bio/taxtriage -r main -latest --outdir test_output -profile <local,docker/singularity>

Modules

0. Subsample (OPTIONAL)
1. Guppyplex (Oxford Nanopore Only)
2. QC Plotting part 1 (pycoQC – Oxford Nanopore)
3. Trimming (Trimgalore – Illumina, Porechop – Oxford Nanopore)
4. Filtering ( Kraken2 – Illlumina, Oxford Nanopore)
5. QC Plotting part 2 (FastQC – Illlumina, Nanoplot – Oxford Nanopore)
6. Classification ( Kraken2 – Illumina, Oxford Nanopore, Krona Plots)
7. Alignment for Stats ( BWAMEM2 – Illumina, Minimap2 – Oxford Nanopore)

• ⚠️Currently, the only realignment is going to be based on a taxid call. For example, if there will not be a complete realignment of "order" despite there being multiple species all within that order. For the most part, this is limited to more specific ranks like species, strain, subspecies etc.

8. Report Generation ( MultiQC – Illumina, Oxford Nanopore)

Credits

TaxTriage was originally written by Brian Merritt, MS Bioinformatics.

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

Contributions and Support

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

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.

Copyright

############################################################################################## Copyright 2022 The Johns Hopkins University Applied Physics Laboratory LLC All rights reserved. Permission is hereby granted, free of charge, to any person obtaining a copy of this software and associated documentation files (the "Software"), to deal in the Software without restriction, including without limitation the rights to use, copy, modify, merge, publish, distribute, sublicense, and/or sell copies of the Software, and to permit persons to whom the Software is furnished to do so.

THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND, EXPRESS OR IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT. IN NO EVENT SHALL THE AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER LIABILITY, WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING FROM, OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN THE SOFTWARE.

Acknowledgements

##############################################################################################

This software tool was supported by the Cooperative Agreement Number NU60OE000104, funded by the Centers for Disease Control and Prevention through the Association of Public Health Laboratories. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the Centers for Disease Control and Prevention, the Department of Health and Human Services, or the Association of Public Health Laboratories.