Test Not Working On Cluster

Introduction

nf-core/viralrecon is a bioinformatics analysis pipeline used to perform assembly and intra-host/low-frequency variant calling for viral samples. The pipeline supports both Illumina and Nanopore sequencing data. For Illumina short-reads the pipeline is able to analyse metagenomics data typically obtained from shotgun sequencing (e.g. directly from clinical samples) and enrichment-based library preparation methods (e.g. amplicon-based: ARTIC SARS-CoV-2 enrichment protocol; or probe-capture-based). For Nanopore data the pipeline only supports amplicon-based analysis obtained from primer sets created and maintained by the ARTIC Network.

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 running the full-sized tests individually for each --platform option can be viewed on the nf-core website and the output directories will be named accordingly i.e. platform_illumina/ and platform_nanopore/.

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!

Pipeline summary

The pipeline has numerous options to allow you to run only specific aspects of the workflow if you so wish. For example, for Illumina data you can skip the host read filtering step with Kraken 2 with --skip_kraken2 or you can skip all of the assembly steps with the --skip_assembly parameter. See the usage and parameter docs for all of the available options when running the pipeline.

The SRA download functionality has been removed from the pipeline (>=2.1) and ported to an independent workflow called nf-core/fetchngs. You can provide --nf_core_pipeline viralrecon when running nf-core/fetchngs to download and auto-create a samplesheet containing publicly available samples that can be accepted directly by the Illumina processing mode of nf-core/viralrecon.

A number of improvements were made to the pipeline recently, mainly with regard to the variant calling. Please see Major updates in v2.3 for a more detailed description.

Illumina

Merge re-sequenced FastQ files (cat)
Read QC (FastQC)
Adapter trimming (fastp)
Removal of host reads (Kraken 2; optional)
Variant calling
1. Read alignment (Bowtie 2)
2. Sort and index alignments (SAMtools)
3. Primer sequence removal (iVar; amplicon data only)
4. Duplicate read marking (picard; optional)
5. Alignment-level QC (picard, SAMtools)
6. Genome-wide and amplicon coverage QC plots (mosdepth)
7. Choice of multiple variant callers (iVar variants; default for amplicon data || BCFTools; default for metagenomics data)
  - Variant annotation (SnpEff, SnpSift)
  - Individual variant screenshots with annotation tracks (ASCIIGenome)
8. Choice of multiple consensus callers (BCFTools, BEDTools; default for both amplicon and metagenomics data || iVar consensus)
  - Consensus assessment report (QUAST)
  - Lineage analysis (Pangolin)
  - Clade assignment, mutation calling and sequence quality checks (Nextclade)
9. Create variants long format table collating per-sample information for individual variants (BCFTools), functional effect prediction (SnpSift) and lineage analysis (Pangolin)
De novo assembly
1. Primer trimming (Cutadapt; amplicon data only)
2. Choice of multiple assembly tools (SPAdes || Unicycler || minia)
  - Blast to reference genome (blastn)
  - Contiguate assembly (ABACAS)
  - Assembly report (PlasmidID)
  - Assembly assessment report (QUAST)
Present QC and visualisation for raw read, alignment, assembly and variant calling results (MultiQC)

Nanopore

Sequencing QC (pycoQC)
Aggregate pre-demultiplexed reads from MinKNOW/Guppy (artic guppyplex)
Read QC (NanoPlot)
Align reads, call variants and generate consensus sequence (artic minion)
Remove unmapped reads and obtain alignment metrics (SAMtools)
Genome-wide and amplicon coverage QC plots (mosdepth)
Downstream variant analysis:
- Count metrics (BCFTools)
- Variant annotation (SnpEff, SnpSift)
- Consensus assessment report (QUAST)
- Lineage analysis (Pangolin)
- Clade assignment, mutation calling and sequence quality checks (Nextclade)
- Individual variant screenshots with annotation tracks (ASCIIGenome)
- Create variants long format table collating per-sample information for individual variants (BCFTools), functional effect prediction (SnpSift) and lineage analysis (Pangolin)
Present QC, visualisation and custom reporting for sequencing, raw reads, alignment and variant calling results (MultiQC)

Quick Start

Install Nextflow (>=22.10.1)
Install any of Docker, Singularity (you can follow this tutorial), Podman, Shifter or Charliecloud for full pipeline reproducibility (you can use Conda both to install Nextflow itself and also to manage software within pipelines. Please only use it within pipelines as a last resort; see docs).
Download the pipeline and test it on a minimal dataset with a single command:
```
nextflow run nf-core/viralrecon -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.
- 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 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.
- If you are using conda, it is highly recommended to use the NXF_CONDA_CACHEDIR or conda.cacheDir settings to store the environments in a central location for future pipeline runs.
Start running your own analysis!
- Please provide pipeline parameters via the CLI 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.
- Typical command for Illumina shotgun analysis:
```
nextflow run nf-core/viralrecon \
    --input samplesheet.csv \
    --outdir <OUTDIR> \
    --platform illumina \
    --protocol metagenomic \
    --genome 'MN908947.3' \
    -profile <docker/singularity/podman/conda/institute>
```
- Typical command for Illumina amplicon analysis:
```
nextflow run nf-core/viralrecon \
    --input samplesheet.csv \
    --outdir <OUTDIR> \
    --platform illumina \
    --protocol amplicon \
    --genome 'MN908947.3' \
    --primer_set artic \
    --primer_set_version 3 \
    --skip_assembly \
    -profile <docker/singularity/podman/conda/institute>
```
- Typical command for Nanopore amplicon analysis:
```
nextflow run nf-core/viralrecon \
    --input samplesheet.csv \
    --outdir <OUTDIR> \
    --platform nanopore \
    --genome 'MN908947.3' \
    --primer_set_version 3 \
    --fastq_dir fastq_pass/ \
    --fast5_dir fast5_pass/ \
    --sequencing_summary sequencing_summary.txt \
    -profile <docker/singularity/podman/conda/institute>
```
- An executable Python script called fastq_dir_to_samplesheet.py has been provided if you are using --platform illumina and would like to auto-create an input samplesheet based on a directory containing FastQ files before you run the pipeline (requires Python 3 installed locally) e.g.
```
wget -L https://raw.githubusercontent.com/nf-core/viralrecon/master/bin/fastq_dir_to_samplesheet.py
./fastq_dir_to_samplesheet.py <FASTQ_DIR> samplesheet.csv
```
- You can find the default keys used to specify --genome in the genomes config file. This provides default options for
  - Reference genomes (including SARS-CoV-2)
  - Genome associates primer sets
  - Nextclade datasets
    
    The Pangolin and Nextclade lineage and clade definitions change regularly as new SARS-CoV-2 lineages are discovered. For instructions to use more recent versions of lineage analysis tools like Pangolin and Nextclade please refer to the updating containers section in the usage docs.
  Where possible we are trying to collate links and settings for standard primer sets to make it easier to run the pipeline with standard keys; see usage docs.

Documentation

The nf-core/viralrecon pipeline comes with documentation about the pipeline usage, parameters and output.

Credits

These scripts were originally written by Sarai Varona, Miguel Juliá, Erika Kvalem and Sara Monzon from BU-ISCIII and co-ordinated by Isabel Cuesta for the Institute of Health Carlos III, Spain. Through collaboration with the nf-core community the pipeline has now been updated substantially to include additional processing steps, to standardise inputs/outputs and to improve pipeline reporting; implemented and maintained primarily by Harshil Patel (@drpatelh) from Seqera Labs, Spain.

The key steps in the Nanopore implementation of the pipeline are carried out using the ARTIC Network's field bioinformatics pipeline and were inspired by the amazing work carried out by contributors to the connor-lab/ncov2019-artic-nf pipeline originally written by Matt Bull for use by the COG-UK project. Thank you for all of your incredible efforts during this pandemic!

Many thanks to others who have helped out and contributed along the way too, including (but not limited to)*:

Name	Affiliation
Aengus Stewart	The Francis Crick Institute, UK
Alexander Peltzer	Boehringer Ingelheim, Germany
Alison Meynert	University of Edinburgh, Scotland
Anthony Underwood	Centre for Genomic Pathogen Surveillance
Anton Korobeynikov	Saint Petersburg State University, Russia
Artem Babaian	University of British Columbia, Canada
Dmitry Meleshko	Saint Petersburg State University, Russia
Edgar Garriga Nogales	Centre for Genomic Regulation, Spain
Erik Garrison	UCSC, USA
Gisela Gabernet	QBiC, University of Tübingen, Germany
Joao Curado	Flomics Biotech, Spain
Jerome Nicod	The Francis Crick Institute, UK
Jose Espinosa-Carrasco	Centre for Genomic Regulation, Spain
Katrin Sameith	DRESDEN-concept Genome Center, Germany
Kevin Menden	QBiC, University of Tübingen, Germany
Lluc Cabus	Flomics Biotech, Spain
Marta Pozuelo	Flomics Biotech, Spain
Maxime Garcia	Seqera Labs, Spain
Michael Heuer	UC Berkeley, USA
Phil Ewels	SciLifeLab, Sweden
Richard Mitter	The Francis Crick Institute, UK
Robert Goldstone	The Francis Crick Institute, UK
Simon Heumos	QBiC, University of Tübingen, Germany
Stephen Kelly	Memorial Sloan Kettering Cancer Center, USA
Thanh Le Viet	Quadram Institute, UK

* Listed in alphabetical order

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 #viralrecon channel (you can join with this invite).

Citations

If you use nf-core/viralrecon for your analysis, please cite it using the following doi: 10.5281/zenodo.3901628

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.

sample	host	Virus sequence	total reads	reads host	% reads host	reads virus	%reads virus	unmapped reads	% unmaped reads	mean DP coverage virus	Coverage > 5x (%)	NumVariantsTrimIVAR	%Nswithoutprimers
201397	human	NC_045512,2	2365486	23052	0,97%	2335037	98,71%	7397	0,3127053	15289,14611	0,985386	5	2.50
201493	human	NC_045512,2	2077038	22299	1,07%	2049058	98,65%	5681	0,2735145	13671,11417	0,997024	6	0.32
201495	human	NC_045512,2	1983106	14431	0,73%	1963048	98,99%	5627	0,28374681	14110,63539	0,997893	7	0.33
201575	human	NC_045512,2	2092372	4073	0,19%	2080486	99,43%	7813	0,37340396	14861,83768	0,998763	7	0.34
201602	human	NC_045512,2	1821320	92766	5,09%	1730140	94,99%	-1586	-0,0870797	11835,88687	0,992308	6	2.27
201607	human	NC_045512,2	2531506	23880	0,94%	2499503	98,74%	8123	0,32087619	18076,30037	0,997458	7	0.49
201617	human	NC_045512,2	2232766	10799	0,48%	2212565	99,10%	9402	0,42109204	16235,09561	0,99786	3	0.32
201706	human	NC_045512,2	2642668	6370	0,24%	2628133	99,45%	8165	0,30896806	18056,17594	0,998763	6	0.24
201709	human	NC_045512,2	1690968	13871	0,82%	1673082	98,94%	4015	0,23743796	11272,06076	0,998595	5	0.64
201738	human	NC_045512,2	2723604	3457	0,13%	2708670	99,45%	11477	0,42139019	18263,53416	0,998763	2	0.32
202050	human	NC_045512,2	1366142	467391	34,21%	898349	65,76%	402	0,02942593	5552,181253	0,95268	5	10.35
202052	human	NC_045512,2	558458	238928	42,78%	167130	29,93%	152400	27,2894291	913,072902	0,946494	4	14.17

	if (params.public_data_ids) {
	include { SRA_DOWNLOAD } from './workflows/sra_download'
	SRA_DOWNLOAD ()

	//
	// WORKFLOW: Variant and de novo assembly analysis for Illumina data
	//
	} else if (params.platform == 'illumina') {
	include { ILLUMINA } from './workflows/illumina'
	ILLUMINA ()

	//
	// WORKFLOW: Variant analysis for Nanopore data
	//
	} else if (params.platform == 'nanopore') {
	include { NANOPORE } from './workflows/nanopore'
	NANOPORE ()
	}

Sample	Nextclade	Pangolin
Frodo	20A	B.1.617.2
Sam	20A	B.1.617.2

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