@ -15,7 +15,8 @@ Learn more about contributing: [CONTRIBUTING.md](https://github.com/nf-core/taxp
- [ ] This comment contains a description of changes (with reason).
- [ ] If you've fixed a bug or added code that should be tested, add tests!
- [ ] If you've added a new tool - have you followed the pipeline conventions in the [contribution docs](https://github.com/nf-core/taxprofiler/tree/master/.github/CONTRIBUTING.md)- [ ] If necessary, also make a PR on the nf-core/taxprofiler _branch_ on the [nf-core/test-datasets](https://github.com/nf-core/test-datasets) repository.
- [ ] If you've added a new tool - have you followed the pipeline conventions in the [contribution docs](https://github.com/nf-core/taxprofiler/tree/master/.github/CONTRIBUTING.md)
- [ ] If necessary, also make a PR on the nf-core/taxprofiler _branch_ on the [nf-core/test-datasets](https://github.com/nf-core/test-datasets) repository.
- [ ] Make sure your code lints (`nf-core lint`).
- [ ] Ensure the test suite passes (`nextflow run . -profile test,docker --outdir <OUTDIR>`).
- [ ] Usage Documentation in `docs/usage.md` is updated.
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The format is based on [Keep a Changelog](https://keepachangelog.com/en/1.0.0/)
and this project adheres to [Semantic Versioning](https://semver.org/spec/v2.0.0.html).
## dev
## v1.1.0dev - [date]
### `Added`
@ -19,6 +19,17 @@ and this project adheres to [Semantic Versioning](https://semver.org/spec/v2.0.0
- [#275](https://github.com/nf-core/taxprofiler/pull/275/files) Replaced function used for error reporting to more Nextflow friendly method (fix by @jfy133)
- [#285](https://github.com/nf-core/taxprofiler/pull/285/files) Fixed overly large log files in Kraken2 output (♥ to @prototaxites for reporting, fix by @Midnighter&@jfy133)
- [#286](https://github.com/nf-core/taxprofiler/pull/286/files) Runtime optimisation of MultiQC step via improved log file processing (fix by @Midnighter&@jfy133)
- [#289](https://github.com/nf-core/taxprofiler/pull/289/files) Pipeline updated to nf-core template 2.8 (fix by @Midnighter&@jfy133)
- [#290](https://github.com/nf-core/taxprofiler/pull/286/files) Minor database input documentation improvements (♥ to @alneberg for reporting, fix by @jfy133)
### `Dependencies`
| Tool | Previous version | New version |
| ------- | ---------------- | ----------- |
| MultiQC | 1.13 | 1.14 |
### `Deprecated`
## v1.0.1 - Dodgy Dachshund Patch [2023-05-15]
### `Added`
@ -27,10 +38,6 @@ and this project adheres to [Semantic Versioning](https://semver.org/spec/v2.0.0
- [#291](https://github.com/nf-core/taxprofiler/pull/291) - Fix Taxpasta not receiving taxonomy directory (❤️ to @SannaAb for reporting, fix by @jfy133)
### `Dependencies`
### `Deprecated`
## v1.0.0 - Dodgy Dachshund [2023-03-13]
Initial release of nf-core/taxprofiler, created with the [nf-core](https://nf-co.re/) template.
[](https://sylabs.io/docs/)
[](https://tower.nf/launch?pipeline=https://github.com/nf-core/taxprofiler)
[](https://nfcore.slack.com/channels/taxprofiler)[](https://twitter.com/nf_core)[](https://www.youtube.com/c/nf-core)
[](https://nfcore.slack.com/channels/taxprofiler)[](https://twitter.com/nf_core)[](https://mstdn.science/@nf_core)[](https://www.youtube.com/c/nf-core)
## Introduction
**nf-core/taxprofiler** is a bioinformatics best-practice analysis pipeline for taxonomic classification and profiling of shotgun metagenomic data. It allows for in-parallel taxonomic identification of reads or taxonomic abundance estimation with multiple classification and profiling tools against multiple databases, produces standardised output tables.
The pipeline is built using [Nextflow](https://www.nextflow.io), 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](https://www.nextflow.io/docs/latest/dsl2.html) 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](https://github.com/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](https://nf-co.re/taxprofiler/results).
The nf-core/taxprofiler CI test dataset uses sequencing data from [Maixner et al. (2021) Curr. Bio.](https://doi.org/10.1016/j.cub.2021.09.031). The AWS full test dataset uses sequencing data and reference genomes from [Meslier (2022) _Sci. Data_](https://doi.org/10.1038/s41597-022-01762-z)
**nf-core/taxprofiler** is a bioinformatics best-practice analysis pipeline for taxonomic classification and profiling of shotgun and long-read metagenomic data. It allows for in-parallel taxonomic identification of reads or taxonomic abundance estimation with multiple classification and profiling tools against multiple databases, produces standardised output tables.
## Pipeline summary
@ -46,43 +40,74 @@ The nf-core/taxprofiler CI test dataset uses sequencing data from [Maixner et al
7. Present QC for raw reads ([`MultiQC`](http://multiqc.info/))
8. Plotting Kraken2, Centrifuge, Kaiju and MALT results ([`Krona`](https://hpc.nih.gov/apps/kronatools.html))
> If you are new to Nextflow and nf-core, please refer to [this page](https://nf-co.re/docs/usage/installation) on how
> to set-up Nextflow. Make sure to [test your setup](https://nf-co.re/docs/usage/introduction#how-to-run-a-pipeline)
> with `-profile test` before running the workflow on actual data.
2. Install any of [`Docker`](https://docs.docker.com/engine/installation/), [`Singularity`](https://www.sylabs.io/guides/3.0/user-guide/) (you can follow [this tutorial](https://singularity-tutorial.github.io/01-installation/)), [`Podman`](https://podman.io/), [`Shifter`](https://nersc.gitlab.io/development/shifter/how-to-use/) or [`Charliecloud`](https://hpc.github.io/charliecloud/) for full pipeline reproducibility _(you can use [`Conda`](https://conda.io/miniconda.html) both to install Nextflow itself and also to manage software within pipelines. Please only use it within pipelines as a last resort; see [docs](https://nf-co.re/usage/configuration#basic-configuration-profiles))_.
First, prepare a samplesheet with your input data that looks as follows:
3. Download the pipeline and test it on a minimal dataset with a single command:
`samplesheet.csv`:
```bash
nextflow run nf-core/taxprofiler -profile test,YOURPROFILE --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.
Each row represents a fastq file (single-end), a pair of fastq files (paired end), or a fasta (with long reads).
> - The pipeline comes with config profiles called `docker`, `singularity`, `podman`, `shifter`, `charliecloud` and `conda` which instruct the pipeline to use the named tool for software management. For example, `-profile test,docker`.
> - Please check [nf-core/configs](https://github.com/nf-core/configs#documentation) 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`](https://nf-co.re/tools/#downloading-pipelines-for-offline-use) command to download images first, before running the pipeline. Setting the [`NXF_SINGULARITY_CACHEDIR` or `singularity.cacheDir`](https://www.nextflow.io/docs/latest/singularity.html?#singularity-docker-hub) Nextflow options enables you to store and re-use the images from a central location for future pipeline runs.
> - If you are using `conda`, it is highly recommended to use the [`NXF_CONDA_CACHEDIR` or `conda.cacheDir`](https://www.nextflow.io/docs/latest/conda.html) settings to store the environments in a central location for future pipeline runs.
Additionally, you will need a database sheet that looks as follows:
That includes directories or `.tar.gz` archives containing databases for the tools you wish to run the pipeline against.
The nf-core/taxprofiler pipeline comes with documentation about the pipeline [usage](https://nf-co.re/taxprofiler/usage), [parameters](https://nf-co.re/taxprofiler/parameters) and [output](https://nf-co.re/taxprofiler/output).
Now, you can run the pipeline using:
## Credits
```bash
nextflow run nf-core/taxprofiler \
-profile <docker/singularity/.../institute> \
--input samplesheet.csv \
--databases databases.csv \
--outdir <OUTDIR> \
--run_kraken2 --run_metaphlan3
```
nf-core/taxprofiler was originally written by [James A. Fellows Yates](https://github.com/jfy133), [Moritz Beber](https://github.com/Midnighter), and [Sofia Stamouli](https://github.com/sofsam).
> **Warning:**
> Please provide pipeline parameters via the CLI (as above) or Nextflow `-params-file` option. Custom config files including those
> provided by the `-c` Nextflow option can be used to provide any configuration _**except for parameters**_;
> see [docs](https://nf-co.re/usage/configuration#custom-configuration-files).
We thank the following people for their contributions to the development of this pipeline:
For more details, please refer to the [usage documentation](https://nf-co.re/taxprofiler/usage) and the [parameter documentation](https://nf-co.re/taxprofiler/parameters).
## Pipeline output
To see the results of a test run with a full size dataset refer to the [results](https://nf-co.re/taxprofiler/results) tab on the nf-core website pipeline page.
For more details about the output files and reports, please refer to the
@ -120,7 +120,7 @@ Column specifications are as follows:
nf-core/taxprofiler will automatically decompress and extract any compressed archives for you.
The (uncompressed) database paths (`db_path`) for each tool are expected to contain the contents of:
The (uncompressed) database paths (`db_path`) for each tool are expected to contain:
- [**Bracken**:](#bracken-custom-database) output of the combined `kraken2-build` and `bracken-build` process.
- [**Centrifuge**:](#centrifuge-custom-database) output of `centrifuge-build`.
@ -130,9 +130,9 @@ The (uncompressed) database paths (`db_path`) for each tool are expected to cont
- [**KrakenUniq**:](#krakenuniq-custom-database) output of `krakenuniq-build` command(s).
- [**MALT**](#malt-custom-database) output of `malt-build`.
- [**MetaPhlAn3**:](#metaphlan3-custom-database) output of with `metaphlan --install` or downloaded from links on the [MetaPhlAn3 wiki](https://github.com/biobakery/MetaPhlAn/wiki/MetaPhlAn-3.0#customizing-the-database).
- [**mOTUs**:](#motus-custom-database) is composed of code and database together.
- [**mOTUs**:](#motus-custom-database) the directory `db_mOTU/` that is downloaded via `motus downloadDB`.
Click the links in the list above for short quick-reference tutorials how to generate custom databases for each tool.
> ℹ️Click the links in the list above for short quick-reference tutorials how to generate custom databases for each tool.
## Running the pipeline
@ -155,6 +155,29 @@ work # Directory containing the nextflow working files
# Other nextflow hidden files, eg. history of pipeline runs and old logs.
```
If you wish to repeatedly use the same parameters for multiple runs, rather than specifying each flag in the command, you can specify these in a params file.
Pipeline settings can be provided in a `yaml` or `json` file via `-params-file <file>`.
> ⚠️ Do not use `-c <file>` to specify parameters as this will result in errors. Custom config files specified with `-c` must only be used for [tuning process resource specifications](https://nf-co.re/docs/usage/configuration#tuning-workflow-resources), other infrastructural tweaks (such as output directories), or module arguments (args).
> The above pipeline run specified with a params file in yaml format:
```bash
nextflow run nf-core/taxprofiler -profile docker -params-file params.yaml
```
with `params.yaml` containing:
```yaml
input: './samplesheet.csv'
outdir: './results/'
genome: 'GRCh37'
input: 'data'
<...>
```
You can also generate such `YAML`/`JSON` files via [nf-core/launch](https://nf-co.re/launch).
### Sequencing quality control
[`FastQC`](https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) gives general quality metrics about your reads. It provides information about the quality score distribution across your reads, per base sequence content (%A/T/G/C), adapter contamination and overrepresented sequences. nf-core taxprofiler offers [`falco`](https://github.com/smithlabcode/falco) as an drop-in replacement, with supposedly better improvement particularly for long reads.
@ -329,6 +352,10 @@ First, go to the [nf-core/taxprofiler releases page](https://github.com/nf-core/
This version number will be logged in reports when you run the pipeline, so that you'll know what you used when you look back in the future. For example, at the bottom of the MultiQC reports.
To further assist in reproducbility, you can use share and re-use [parameter files](#running-the-pipeline) to repeat pipeline runs with the same settings without having to write out a command with every single parameter.
> 💡 If you wish to share such profile (such as upload as supplementary material for academic publications), make sure to NOT include cluster specific paths to files, nor institutional specific profiles.
## Core Nextflow arguments
> **NB:** These options are part of Nextflow and use a _single_ hyphen (pipeline parameters use a double-hyphen).
@ -337,7 +364,7 @@ This version number will be logged in reports when you run the pipeline, so that
Use this parameter to choose a configuration profile. Profiles can give configuration presets for different compute environments.
Several generic profiles are bundled with the pipeline which instruct the pipeline to use software packaged using different methods (Docker, Singularity, Podman, Shifter, Charliecloud, Conda) - see below.
Several generic profiles are bundled with the pipeline which instruct the pipeline to use software packaged using different methods (Docker, Singularity, Podman, Shifter, Charliecloud, Apptainer, Conda) - see below.
> We highly recommend the use of Docker or Singularity containers for full pipeline reproducibility, however when this is not possible, Conda is also supported.
@ -361,8 +388,10 @@ If `-profile` is not specified, the pipeline will run locally and expect all sof
- A generic configuration profile to be used with [Shifter](https://nersc.gitlab.io/development/shifter/how-to-use/)
- `charliecloud`
- A generic configuration profile to be used with [Charliecloud](https://hpc.github.io/charliecloud/)
- `apptainer`
- A generic configuration profile to be used with [Apptainer](https://apptainer.org/)
- `conda`
- A generic configuration profile to be used with [Conda](https://conda.io/docs/). Please only use Conda as a last resort i.e. when it's not possible to run the pipeline with Docker, Singularity, Podman, Shifter or Charliecloud.
- A generic configuration profile to be used with [Conda](https://conda.io/docs/). Please only use Conda as a last resort i.e. when it's not possible to run the pipeline with Docker, Singularity, Podman, Shifter, Charliecloud, or Apptainer.
### `-resume`
@ -380,102 +409,19 @@ Specify the path to a specific config file (this is a core Nextflow command). Se
Whilst the default requirements set within the pipeline will hopefully work for most people and with most input data, you may find that you want to customise the compute resources that the pipeline requests. Each step in the pipeline has a default set of requirements for number of CPUs, memory and time. For most of the steps in the pipeline, if the job exits with any of the error codes specified [here](https://github.com/nf-core/rnaseq/blob/4c27ef5610c87db00c3c5a3eed10b1d161abf575/conf/base.config#L18) it will automatically be resubmitted with higher requests (2 x original, then 3 x original). If it still fails after the third attempt then the pipeline execution is stopped.
For example, if the nf-core/rnaseq pipeline is failing after multiple re-submissions of the `STAR_ALIGN` process due to an exit code of `137` this would indicate that there is an out of memory issue:
```console
[62/149eb0] NOTE: Process `NFCORE_RNASEQ:RNASEQ:ALIGN_STAR:STAR_ALIGN (WT_REP1)` terminated with an error exit status (137) -- Execution is retried (1)
Error executing process > 'NFCORE_RNASEQ:RNASEQ:ALIGN_STAR:STAR_ALIGN (WT_REP1)'
Caused by:
Process `NFCORE_RNASEQ:RNASEQ:ALIGN_STAR:STAR_ALIGN (WT_REP1)` terminated with an error exit status (137)
Command executed:
STAR \
--genomeDir star \
--readFilesIn WT_REP1_trimmed.fq.gz \
--runThreadN 2 \
--outFileNamePrefix WT_REP1. \
<TRUNCATED>
Command exit status:
137
Command output:
(empty)
Command error:
.command.sh: line 9: 30 Killed STAR --genomeDir star --readFilesIn WT_REP1_trimmed.fq.gz --runThreadN 2 --outFileNamePrefix WT_REP1. <TRUNCATED>
Tip: you can replicate the issue by changing to the process work dir and entering the command `bash .command.run`
```
#### For beginners
A first step to bypass this error, you could try to increase the amount of CPUs, memory, and time for the whole pipeline. Therefor you can try to increase the resource for the parameters `--max_cpus`, `--max_memory`, and `--max_time`. Based on the error above, you have to increase the amount of memory. Therefore you can go to the [parameter documentation of rnaseq](https://nf-co.re/rnaseq/3.9/parameters) and scroll down to the `show hidden parameter` button to get the default value for `--max_memory`. In this case 128GB, you than can try to run your pipeline again with `--max_memory 200GB -resume` to skip all process, that were already calculated. If you can not increase the resource of the complete pipeline, you can try to adapt the resource for a single process as mentioned below.
#### Advanced option on process level
To bypass this error you would need to find exactly which resources are set by the `STAR_ALIGN` process. The quickest way is to search for `process STAR_ALIGN` in the [nf-core/rnaseq Github repo](https://github.com/nf-core/rnaseq/search?q=process+STAR_ALIGN).
We have standardised the structure of Nextflow DSL2 pipelines such that all module files will be present in the `modules/` directory and so, based on the search results, the file we want is `modules/nf-core/star/align/main.nf`.
If you click on the link to that file you will notice that there is a `label` directive at the top of the module that is set to [`label process_high`](https://github.com/nf-core/rnaseq/blob/4c27ef5610c87db00c3c5a3eed10b1d161abf575/modules/nf-core/software/star/align/main.nf#L9).
The [Nextflow `label`](https://www.nextflow.io/docs/latest/process.html#label) directive allows us to organise workflow processes in separate groups which can be referenced in a configuration file to select and configure subset of processes having similar computing requirements.
The default values for the `process_high` label are set in the pipeline's [`base.config`](https://github.com/nf-core/rnaseq/blob/4c27ef5610c87db00c3c5a3eed10b1d161abf575/conf/base.config#L33-L37) which in this case is defined as 72GB.
Providing you haven't set any other standard nf-core parameters to **cap** the [maximum resources](https://nf-co.re/usage/configuration#max-resources) used by the pipeline then we can try and bypass the `STAR_ALIGN` process failure by creating a custom config file that sets at least 72GB of memory, in this case increased to 100GB.
The custom config below can then be provided to the pipeline via the [`-c`](#-c) parameter as highlighted in previous sections.
> **NB:** We specify the full process name i.e. `NFCORE_RNASEQ:RNASEQ:ALIGN_STAR:STAR_ALIGN` in the config file because this takes priority over the short name (`STAR_ALIGN`) and allows existing configuration using the full process name to be correctly overridden.
>
> If you get a warning suggesting that the process selector isn't recognised check that the process name has been specified correctly.
### Updating containers (advanced users)
The [Nextflow DSL2](https://www.nextflow.io/docs/latest/dsl2.html) implementation of this pipeline uses one container per process which makes it much easier to maintain and update software dependencies. If for some reason you need to use a different version of a particular tool with the pipeline then you just need to identify the `process` name and override the Nextflow `container` definition for that process using the `withName` declaration. For example, in the [nf-core/viralrecon](https://nf-co.re/viralrecon) pipeline a tool called [Pangolin](https://github.com/cov-lineages/pangolin) has been used during the COVID-19 pandemic to assign lineages to SARS-CoV-2 genome sequenced samples. Given that the lineage assignments change quite frequently it doesn't make sense to re-release the nf-core/viralrecon everytime a new version of Pangolin has been released. However, you can override the default container used by the pipeline by creating a custom config file and passing it as a command-line argument via `-c custom.config`.
1. Check the default version used by the pipeline in the module file for [Pangolin](https://github.com/nf-core/viralrecon/blob/a85d5969f9025409e3618d6c280ef15ce417df65/modules/nf-core/software/pangolin/main.nf#L14-L19)
2. Find the latest version of the Biocontainer available on [Quay.io](https://quay.io/repository/biocontainers/pangolin?tag=latest&tab=tags)
3. Create the custom config accordingly:
- For Docker:
To change the resource requests, please see the [max resources](https://nf-co.re/docs/usage/configuration#max-resources) and [tuning workflow resources](https://nf-co.re/docs/usage/configuration#tuning-workflow-resources) section of the nf-core website.
In some cases you may wish to change which container or conda environment a step of the pipeline uses for a particular tool. By default nf-core pipelines use containers and software from the [biocontainers](https://biocontainers.pro/) or [bioconda](https://bioconda.github.io/) projects. However in some cases the pipeline specified version maybe out of date.
To use a different container from the default container or conda environment specified in a pipeline, please see the [updating tool versions](https://nf-co.re/docs/usage/configuration#updating-tool-versions) section of the nf-core website.
- For Conda:
### Custom Tool Arguments
```nextflow
process {
withName: PANGOLIN {
conda = 'bioconda::pangolin=3.0.5'
}
}
```
A pipeline might not always support every possible argument or option of a particular tool used in pipeline. Fortunately, nf-core pipelines provide some freedom to users to insert additional parameters that the pipeline does not include by default.
> **NB:** If you wish to periodically update individual tool-specific results (e.g. Pangolin) generated by the pipeline then you must ensure to keep the `work/` directory otherwise the `-resume` ability of the pipeline will be compromised and it will restart from scratch.
To learn how to provide additional arguments to a particular tool of the pipeline, please see the [customising tool arguments](https://nf-co.re/docs/usage/configuration#customising-tool-arguments) section of the nf-core website.
James A. Fellows Yates
12 months ago
committed by