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A mapping-based pipeline for creating a phylogeny from bacterial whole genome sequences

Home Page: https://nf-co.re/bactmap

License: MIT License

HTML 1.42% Python 7.44% Nextflow 68.02% Groovy 23.12%

nf-core nextflow workflow pipeline bacteria bacterial bacterial-genome-analysis phylogeny mapping genomics

bactmap's Introduction

Introduction

nf-core/bactmap is a bioinformatics best-practice analysis pipeline for mapping short reads from bacterial WGS to a reference sequence, creating filtered VCF files, making pseudogenomes based on high quality positions in the VCF files and optionally creating a phylogeny from an alignment of the pseudogenomes.

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.

Pipeline summary

The pipeline is composed of the following steps:

Index reference fasta file (BWA index)
Trim reads for quality and adapter sequence (Optional) (fastp)
Estimate genome size (mash sketch)
Downsample fastq files (Optional) (Rasusa)
Variant calling
1. Read mapping (BWA mem)
2. Sort and index alignments (SAMtools)
3. Call and filter variants (BCFtools)
4. Convert filtered bcf to pseudogenome fasta (vcf2pseudogenome.py)
Create alignment from pseudogenome by concatenating fasta files having first checked that the sample sequences are high quality(calculate_fraction_of_non_GATC_bases.py)
Remove recombination (Optional) (Gubbins)
Extract variant sites from alignment (SNP-sites)
Construct phylogenetic tree (Optional)
1. Fast/less accurate
  - neighbour joining RapidNJ
  - approximate maximum likelihood FastTree2)
2. Slow/more accurate, maximum likelihood
  - IQ-TREE,
  - RAxML-NG

Quick Start

Install Nextflow (>=21.04.0)
Install any of Docker, Singularity, Podman, Shifter or Charliecloud for full pipeline reproducibility (please only use Conda as a last resort; see docs)
Download the pipeline and test it on a minimal dataset with a single command:
```
nextflow run nf-core/bactmap -profile test,<docker/singularity/podman/shifter/charliecloud/conda/institute>
```
- 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 then the pipeline will auto-detect this and attempt to download the Singularity images directly as opposed to performing a conversion from Docker images. If you are persistently observing issues downloading Singularity images directly due to timeout or network issues then please use the --singularity_pull_docker_container parameter to pull and convert the Docker image instead. Alternatively, it is highly recommended to use the nf-core download command to pre-download all of the required containers before running the pipeline and to set the NXF_SINGULARITY_CACHEDIR or singularity.cacheDir Nextflow options to be able 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!

nextflow run nf-core/bactmap -profile <docker/singularity/podman/conda/institute> --input samplesheet.csv --reference chromosome.fasta

Documentation

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

Credits

nf-core/bactmap was originally written by Anthony Underwood, Andries van Tonder and Thanh Le Viet.

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

Anthony Underwood's time working on the project was funded by the National Institute for Health Research (NIHR) Global Health Research Unit for the Surveillance of Antimicrobial Resistance (Grant Reference Number 16/136/111)

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 #bactmap 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.

bactmap's People

Contributors

Stargazers

Watchers

bactmap's Issues

option to keep the trimmed reads

download reads from the SRA/ENA

Add pseudogenome count check before running gubbins

rapidnj DSL2 module

calculation of snps to exclude LowQual positions

Is your feature request related to a problem? Please describe

The total number of snp differences reported in the output multiqc plot includes low quality snps.

Sample Name	SNP
Leg180-11142022-KCH-D5512A	15

#CHROM	POS	QUAL	FILTER
DACUAB010000005.1	133396	228	PASS
DACUAB010000005.1	133545	228	LowQual
DACUAB010000005.1	133669	228	PASS
DACUAB010000011.1	24	8.99921	LowQual
DACUAB010000011.1	27	8.99921	LowQual
DACUAB010000011.1	51	7.30814	LowQual
DACUAB010000011.1	54	7.30814	LowQual
DACUAB010000018.1	45824	30.4183	LowQual
DACUAB010000025.1	50907	6.51248	LowQual
DACUAB010000030.1	203	111	LowQual
DACUAB010000068.1	39	164	LowQual
DACUAB010000068.1	44	170	LowQual
DACUAB010000068.1	45	166	LowQual
DACUAB010000068.1	46	163	LowQual
DACUAB010000068.1	50	161	LowQual

Describe the solution you'd like

Can a second column in the multiqc plot include the number of snps that pass quality filters?
One possible option would be to use the --apply-filters flag (see below) but I have not confirmed what all other information gets used from the bcftools stats command in the multiqc plot.
bcftools stats -f PASS test.filtered.vcf.gz

So instead of the 15 reported snps reported in the multiqc plot, by using the filter function, the reported number of snps would be indicative of those that pass the bcftools filters that are applied (see below).

Sample Name	SNP
Leg180-11142022-KCH-D5512A	2

But maybe this is not an issue for others, so unclear how much adding this feature is helpful for larger community.

Describe alternatives you've considered

At present, I only discovered this by visually examining some output.

Additional context

N/A

rasusa DSL2 module

https://github.com/mbhall88/rasusa

See nf-core/modules#413

Cannot get property 'args' on null object error

Hi,
Thanks for building this pipeline! I'm running into an error that I haven't been able to solve, copied below. Thanks in advance!

command: nextflow run nf-core/bactmap -profile test,singularity

error seen:

Cannot get property 'args' on null object
 -- Check script '/home/kcreamer/.nextflow/assets/nf-core/bactmap/./workflows/bactmap.nf' at line: 38 or see '.nextflow.log' file for more details

Line 38 in the script I believe is:
multiqc_options.args += params.multiqc_title ? Utils.joinModuleArgs(["--title \"$params.multiqc_title\""]) : ''

(I thus tried running the same command but with multiqc turned off, ie nextflow run nf-core/bactmap -profile test,singularity --skip_multiqc but the same null args error occurred).

And the info in the .nextflow.log reads:

May-10 13:29:18.605 [main] DEBUG nextflow.Session - Session aborted -- Cause: Cannot get property 'args' on null object
May-10 13:29:18.637 [main] ERROR nextflow.cli.Launcher - @unknown
java.lang.NullPointerException: Cannot get property 'args' on null object
        at org.codehaus.groovy.runtime.NullObject.getProperty(NullObject.java:60)
        at org.codehaus.groovy.runtime.InvokerHelper.getProperty(InvokerHelper.java:194)
        at org.codehaus.groovy.runtime.callsite.NullCallSite.getProperty(NullCallSite.java:46)
        at org.codehaus.groovy.runtime.callsite.AbstractCallSite.callGetProperty(AbstractCallSite.java:329)
        at Script_c99ee09b.runScript(Script_c99ee09b:38)
        at nextflow.script.BaseScript.runDsl2(BaseScript.groovy:170)
        at nextflow.script.BaseScript.run(BaseScript.groovy:203)
        at nextflow.script.ScriptParser.runScript(ScriptParser.groovy:220)
        at nextflow.script.ScriptParser.runScript(ScriptParser.groovy:206)
        at nextflow.script.ScriptParser.runScript(ScriptParser.groovy:200)
        at nextflow.script.IncludeDef.memoizedMethodPriv$loadModule0PathMapSession(IncludeDef.groovy:142)
        at nextflow.script.IncludeDef.access$0(IncludeDef.groovy)
        at nextflow.script.IncludeDef$__clinit__closure1.doCall(IncludeDef.groovy)
        at java.base/jdk.internal.reflect.NativeMethodAccessorImpl.invoke0(Native Method)
        at java.base/jdk.internal.reflect.NativeMethodAccessorImpl.invoke(NativeMethodAccessorImpl.java:62)
        at java.base/jdk.internal.reflect.DelegatingMethodAccessorImpl.invoke(DelegatingMethodAccessorImpl.java:43)
        at java.base/java.lang.reflect.Method.invoke(Method.java:566)
        at org.codehaus.groovy.reflection.CachedMethod.invoke(CachedMethod.java:107)
        at groovy.lang.MetaMethod.doMethodInvoke(MetaMethod.java:323)
        at org.codehaus.groovy.runtime.metaclass.ClosureMetaClass.invokeMethod(ClosureMetaClass.java:274)
        at groovy.lang.MetaClassImpl.invokeMethod(MetaClassImpl.java:1035)
        at groovy.lang.Closure.call(Closure.java:412)
        at org.codehaus.groovy.runtime.memoize.Memoize$MemoizeFunction.lambda$call$0(Memoize.java:137)
        at org.codehaus.groovy.runtime.memoize.ConcurrentCommonCache.getAndPut(ConcurrentCommonCache.java:137)
        at org.codehaus.groovy.runtime.memoize.ConcurrentCommonCache.getAndPut(ConcurrentCommonCache.java:113)
        at org.codehaus.groovy.runtime.memoize.Memoize$MemoizeFunction.call(Memoize.java:136)
        at nextflow.script.IncludeDef.loadModule0(IncludeDef.groovy)
        at nextflow.script.IncludeDef.load0(IncludeDef.groovy:114)
        at nextflow.script.IncludeDef$load0$1.call(Unknown Source)
        at org.codehaus.groovy.runtime.callsite.CallSiteArray.defaultCall(CallSiteArray.java:47)
        at org.codehaus.groovy.runtime.callsite.AbstractCallSite.call(AbstractCallSite.java:125)
        at org.codehaus.groovy.runtime.callsite.AbstractCallSite.call(AbstractCallSite.java:139)
        at Script_14934cd2.runScript(Script_14934cd2:28)
        at nextflow.script.BaseScript.runDsl2(BaseScript.groovy:170)
        at nextflow.script.BaseScript.run(BaseScript.groovy:203)
        at nextflow.script.ScriptParser.runScript(ScriptParser.groovy:220)
        at nextflow.script.ScriptRunner.run(ScriptRunner.groovy:212)
        at nextflow.script.ScriptRunner.execute(ScriptRunner.groovy:120)
        at nextflow.cli.CmdRun.run(CmdRun.groovy:334)
        at nextflow.cli.Launcher.run(Launcher.groovy:480)
        at nextflow.cli.Launcher.main(Launcher.groovy:639

I tried running another nf-core pipeline (nf-core/mag) and it got past this step & didn't return the same "null args" error, so it seems so far to be specific to this pipeline.

Specs:

Nextflow Version: 22.04.0
SingularityPRO version 3.5-8.el7
System: Linux 3.10.0-1160.45.1.el7.x86_64
Runtime: Groovy 3.0.10 on OpenJDK 64-Bit Server VM 11.0.9.1-internal+0-adhoc..src
nf-core/bactmap v1.0.0
Nextflow was called from an activated conda environment; singularity was also pre-loaded.

Thanks in advance for your help with this!

Modules required for DSL 2 implementation of pipeline

fasttree DSL2 module

iqtree DSL2 module

seqtk sample DLS2 module

mash sketch DSL2 module

Test docker

raxml-ng DSL2 module

Test singularity

bcftools call DSL2 module

gubbins DSL2 module

pipeline fails when parameter trim = false

Check Documentation

I have checked the following places for your error:

Description of the bug

When setting trim = false, the pipeline stops with the error:

Jun-27 17:04:36.559 [main] ERROR nextflow.cli.Launcher - Access to 'FASTP.out' is undefined since the process 'FASTP' has not been invoked before accessing the output attribute
nextflow.exception.ScriptRuntimeException: Access to 'FASTP.out' is undefined since the process 'FASTP' has not been invoked before accessing the output attribute

Steps to reproduce

Steps to reproduce the behaviour:

nextflow run nf-core/bactmap -profile singularity
nextflow.config

...
    trim       = false
...

See error: as above

Expected behaviour

When using already trimmed samples, we should be able to run bactmap with trim = false

Log files

Have you provided the following extra information/files:

The command used to run the pipeline

#!/bin/bash -l
#PBS -N bactmaptest
#PBS -l select=1:ncpus=2:mem=5gb
#PBS -l walltime=168:00:00

cd $PBS_O_WORKDIR
module load java
NXF_OPTS='-Xms1g -Xmx4g'

nextflow run nf-core/bactmap -profile singularity

nextflow.config

params {
    max_memory = '2.GB'
    max_cpus   = 2
    max_time   = '1.h'
    rapidnj    = true
    fasttree   = true
    iqtree     = true
    raxmlng    = true
    trim       = false
    input      = 'samplesheet.csv'
    reference  = 'NCTC13799.fna'
}

The .nextflow.log file

Jun-27 17:04:21.420 [main] DEBUG nextflow.cli.Launcher - $> nextflow run nf-core/bactmap -profile singularity
Jun-27 17:04:21.547 [main] INFO  nextflow.cli.CmdRun - N E X T F L O W  ~  version 22.04.4
Jun-27 17:04:23.140 [main] DEBUG nextflow.scm.AssetManager - Git config: /home/windell/.nextflow/assets/nf-core/bactmap/.git/config; branch: master; remote: origin; url: https://github.com/nf-core/bactmap.git
Jun-27 17:04:23.161 [main] DEBUG nextflow.scm.AssetManager - Git config: /home/windell/.nextflow/assets/nf-core/bactmap/.git/config; branch: master; remote: origin; url: https://github.com/nf-core/bactmap.git
Jun-27 17:04:24.570 [main] DEBUG nextflow.config.ConfigBuilder - Found config home: /home/windell/.nextflow/config
Jun-27 17:04:24.570 [main] DEBUG nextflow.config.ConfigBuilder - Found config base: /home/windell/.nextflow/assets/nf-core/bactmap/nextflow.config
Jun-27 17:04:24.572 [main] DEBUG nextflow.config.ConfigBuilder - Found config local: /mnt/work/eresearch_bio/nextflow/nf-core/bactmap/testdata/run01/nextflow.config
Jun-27 17:04:24.573 [main] DEBUG nextflow.config.ConfigBuilder - Parsing config file: /home/windell/.nextflow/config
Jun-27 17:04:24.573 [main] DEBUG nextflow.config.ConfigBuilder - Parsing config file: /home/windell/.nextflow/assets/nf-core/bactmap/nextflow.config
Jun-27 17:04:24.573 [main] DEBUG nextflow.config.ConfigBuilder - Parsing config file: /mnt/work/eresearch_bio/nextflow/nf-core/bactmap/testdata/run01/nextflow.config
Jun-27 17:04:24.579 [main] DEBUG nextflow.config.ConfigBuilder - Applying config profile: `singularity`
Jun-27 17:04:24.671 [main] DEBUG nextflow.config.ConfigBuilder - Applying config profile: `singularity`
Jun-27 17:04:25.275 [main] DEBUG nextflow.plugin.PluginsFacade - Using Default plugins manager
Jun-27 17:04:25.291 [main] INFO  org.pf4j.DefaultPluginStatusProvider - Enabled plugins: []
Jun-27 17:04:25.292 [main] INFO  org.pf4j.DefaultPluginStatusProvider - Disabled plugins: []
Jun-27 17:04:25.295 [main] INFO  org.pf4j.DefaultPluginManager - PF4J version 3.4.1 in 'deployment' mode
Jun-27 17:04:25.312 [main] DEBUG nextflow.plugin.PluginsFacade - Using Default plugins manager
Jun-27 17:04:25.611 [main] DEBUG nextflow.config.ConfigBuilder - Applying config profile: `singularity`
Jun-27 17:04:25.652 [main] DEBUG nextflow.config.ConfigBuilder - Available config profiles: [cfc_dev, ifb_core, denbi_qbic, genotoul, alice, mjolnir_globe, uppmax, abims, nihbiowulf, nu_genomics, oist, sahmri, mpcdf, leicester, lugh, vsc_ugent, sage, cambridge, unibe_ibu, vai, podman, czbiohub_aws, jax, cheaha, ccga_med, test, google, computerome, seg_globe, sanger, pasteur, test_full, eddie, azurebatch, bi, bigpurple, cedars, docker, gis, eva, utd_ganymede, charliecloud, fgcz, conda, singularity, icr_davros, munin, rosalind, prince, hasta, hebbe, cfc, utd_sysbio, uzh, debug, genouest, cbe, ebc, ccga_dx, crick, marvin, phoenix, biohpc_gen, shifter, awsbatch, uct_hpc, imperial, maestro, aws_tower, binac]
Jun-27 17:04:25.721 [main] DEBUG nextflow.cli.CmdRun - Applied DSL=2 from script declararion
Jun-27 17:04:25.722 [main] INFO  nextflow.cli.CmdRun - Launching `https://github.com/nf-core/bactmap` [peaceful_bhaskara] DSL2 - revision: e83f8c5f0e [master]
Jun-27 17:04:25.723 [main] DEBUG nextflow.plugin.PluginsFacade - Setting up plugin manager > mode=prod; plugins-dir=/home/windell/.nextflow/plugins; core-plugins: [email protected],[email protected],[email protected],[email protected],[email protected],[email protected],[email protected]
Jun-27 17:04:25.727 [main] DEBUG nextflow.plugin.PluginsFacade - Plugins default=[]
Jun-27 17:04:25.768 [main] INFO  org.pf4j.DefaultPluginStatusProvider - Enabled plugins: []
Jun-27 17:04:25.768 [main] INFO  org.pf4j.DefaultPluginStatusProvider - Disabled plugins: []
Jun-27 17:04:25.770 [main] INFO  org.pf4j.DefaultPluginManager - PF4J version 3.4.1 in 'deployment' mode
Jun-27 17:04:25.780 [main] INFO  org.pf4j.AbstractPluginManager - No plugins
Jun-27 17:04:25.780 [main] DEBUG nextflow.plugin.PluginUpdater - Installing plugin nf-tower version: 1.4.0
Jun-27 17:04:25.808 [main] INFO  org.pf4j.AbstractPluginManager - Plugin '[email protected]' resolved
Jun-27 17:04:25.808 [main] INFO  org.pf4j.AbstractPluginManager - Start plugin '[email protected]'
Jun-27 17:04:26.133 [main] DEBUG nextflow.plugin.BasePlugin - Plugin started [email protected]
Jun-27 17:04:26.278 [main] DEBUG nextflow.Session - Session uuid: 102eada9-1cd2-4eab-99eb-61fa2212febc
Jun-27 17:04:26.278 [main] DEBUG nextflow.Session - Run name: peaceful_bhaskara
Jun-27 17:04:26.279 [main] DEBUG nextflow.Session - Executor pool size: 2
Jun-27 17:04:26.293 [main] DEBUG nextflow.config.ConfigBuilder - Found config home: /home/windell/.nextflow/config
Jun-27 17:04:26.293 [main] DEBUG nextflow.config.ConfigBuilder - Found config base: /home/windell/.nextflow/assets/nf-core/bactmap/nextflow.config
Jun-27 17:04:26.293 [main] DEBUG nextflow.config.ConfigBuilder - Found config local: /mnt/work/eresearch_bio/nextflow/nf-core/bactmap/testdata/run01/nextflow.config
Jun-27 17:04:26.293 [main] DEBUG nextflow.config.ConfigBuilder - Parsing config file: /home/windell/.nextflow/config
Jun-27 17:04:26.293 [main] DEBUG nextflow.config.ConfigBuilder - Parsing config file: /home/windell/.nextflow/assets/nf-core/bactmap/nextflow.config
Jun-27 17:04:26.293 [main] DEBUG nextflow.config.ConfigBuilder - Parsing config file: /mnt/work/eresearch_bio/nextflow/nf-core/bactmap/testdata/run01/nextflow.config
Jun-27 17:04:26.294 [main] DEBUG nextflow.config.ConfigBuilder - Applying config profile: `singularity`
Jun-27 17:04:26.346 [main] DEBUG nextflow.config.ConfigBuilder - Applying config profile: `singularity`
Jun-27 17:04:26.779 [main] DEBUG nextflow.config.ConfigBuilder - Applying config profile: `singularity`
Jun-27 17:04:26.803 [main] DEBUG nextflow.config.ConfigBuilder - Available config profiles: [cfc_dev, ifb_core, denbi_qbic, genotoul, alice, mjolnir_globe, uppmax, abims, nihbiowulf, nu_genomics, oist, sahmri, mpcdf, leicester, lugh, vsc_ugent, sage, cambridge, unibe_ibu, vai, podman, czbiohub_aws, jax, cheaha, ccga_med, test, google, computerome, seg_globe, sanger, pasteur, test_full, eddie, azurebatch, bi, bigpurple, cedars, docker, gis, eva, utd_ganymede, charliecloud, fgcz, conda, singularity, icr_davros, munin, rosalind, prince, hasta, hebbe, cfc, utd_sysbio, uzh, debug, genouest, cbe, ebc, ccga_dx, crick, marvin, phoenix, biohpc_gen, shifter, awsbatch, uct_hpc, imperial, maestro, aws_tower, binac]
Jun-27 17:04:26.852 [main] DEBUG nextflow.cli.CmdRun -
  Version: 22.04.4 build 5706
  Created: 19-06-2022 20:51 UTC (20-06-2022 06:51 AEDT)
  System: Linux 4.12.14-122.121-default
  Runtime: Groovy 3.0.10 on OpenJDK 64-Bit Server VM 11.0.2+9
  Encoding: UTF-8 (UTF-8)
  Process: 7733@cl4n058 [10.13.1.8]
  CPUs: 2 - Mem: 377.2 GB (154.8 GB) - Swap: 2 GB (1.8 GB)
Jun-27 17:04:26.914 [main] DEBUG nextflow.Session - Work-dir: /mnt/work/eresearch_bio/nextflow/nf-core/bactmap/testdata/run01/work [nfs]
Jun-27 17:04:26.935 [main] DEBUG nextflow.executor.ExecutorFactory - Extension executors providers=[]
Jun-27 17:04:26.949 [main] DEBUG nextflow.Session - Observer factory: DefaultObserverFactory
Jun-27 17:04:26.975 [main] DEBUG nextflow.Session - Observer factory: TowerFactory
Jun-27 17:04:27.491 [main] DEBUG nextflow.cache.CacheFactory - Using Nextflow cache factory: nextflow.cache.DefaultCacheFactory
Jun-27 17:04:27.513 [main] DEBUG nextflow.util.CustomThreadPool - Creating default thread pool > poolSize: 3; maxThreads: 1000
Jun-27 17:04:27.782 [main] DEBUG nextflow.Session - Session start invoked
Jun-27 17:04:27.786 [main] DEBUG nextflow.trace.TraceFileObserver - Flow starting -- trace file: /mnt/work/eresearch_bio/nextflow/nf-core/bactmap/testdata/run01/results/pipeline_info/execution_trace_2022-06-27_17-04-25.txt
Jun-27 17:04:27.875 [main] DEBUG io.seqera.tower.plugin.TowerClient - Creating Tower observer -- endpoint=https://nftower.qut.edu.au/api; requestInterval=1s; aliveInterval=1m; maxRetries=5; backOffBase=3; backOffDelay=250
Jun-27 17:04:27.997 [main] DEBUG nextflow.Session - Using default localLib path: /home/windell/.nextflow/assets/nf-core/bactmap/lib
Jun-27 17:04:28.008 [main] DEBUG nextflow.Session - Adding to the classpath library: /home/windell/.nextflow/assets/nf-core/bactmap/lib
Jun-27 17:04:28.009 [main] DEBUG nextflow.Session - Adding to the classpath library: /home/windell/.nextflow/assets/nf-core/bactmap/lib/nfcore_external_java_deps.jar
Jun-27 17:04:29.524 [main] DEBUG nextflow.script.ScriptRunner - > Launching execution
Jun-27 17:04:29.931 [main] INFO  nextflow.Nextflow -

------------------------------------------------------
                                        ,--./,-.
        ___     __   __   __   ___     /,-._.--~'
  |\ | |__  __ /  ` /  \ |__) |__         }  {
  | \| |       \__, \__/ |  \ |___     \`-._,-`-,
                                        `._,._,'
  nf-core/bactmap v1.0.0
------------------------------------------------------
Core Nextflow options
  revision       : master
  runName        : peaceful_bhaskara
  containerEngine: singularity
  launchDir      : /mnt/work/eresearch_bio/nextflow/nf-core/bactmap/testdata/run01
  workDir        : /mnt/work/eresearch_bio/nextflow/nf-core/bactmap/testdata/run01/work
  projectDir     : /home/windell/.nextflow/assets/nf-core/bactmap
  userName       : windell
  profile        : singularity
  configFiles    : /home/windell/.nextflow/config, /home/windell/.nextflow/assets/nf-core/bactmap/nextflow.config, /mnt/work/eresearch_bio/nextflow/nf-core/bactmap/testdata/run01/nextflow.config

Input/output options
  input          : samplesheet.csv

Compulsory parameters
  reference      : NCTC13799.fna

Optional pipeline steps
  trim           : false
  adapter_file   : /home/windell/.nextflow/assets/nf-core/bactmap/assets/adapters.fas
  rapidnj        : true
  fasttree       : true
  iqtree         : true
  raxmlng        : true

Max job request options
  max_cpus       : 2
  max_memory     : 2.GB
  max_time       : 1.h

!! Only displaying parameters that differ from the pipeline defaults !!
------------------------------------------------------
If you use nf-core/bactmap for your analysis please cite:

* The nf-core framework
  https://doi.org/10.1038/s41587-020-0439-x

* Software dependencies
  https://github.com/nf-core/bactmap/blob/master/CITATIONS.md
------------------------------------------------------
Jun-27 17:04:35.850 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: pbspro
Jun-27 17:04:35.850 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'pbspro'
Jun-27 17:04:35.860 [main] DEBUG nextflow.executor.Executor - [warm up] executor > pbspro
Jun-27 17:04:35.865 [main] DEBUG n.processor.TaskPollingMonitor - Creating task monitor for executor 'pbspro' > capacity: 100; pollInterval: 5s; dumpInterval: 5m
Jun-27 17:04:35.869 [main] DEBUG n.executor.AbstractGridExecutor - Creating executor 'pbspro' > queue-stat-interval: 1m
Jun-27 17:04:36.035 [main] DEBUG nextflow.script.ProcessConfig - Config settings `withLabel:process_high` matches labels `process_high` for process with name NFCORE_BACTMAP:BACTMAP:BWA_INDEX
Jun-27 17:04:36.035 [main] DEBUG nextflow.script.ProcessConfig - Config settings `withName:BWA_INDEX` matches process NFCORE_BACTMAP:BACTMAP:BWA_INDEX
Jun-27 17:04:36.048 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: pbspro
Jun-27 17:04:36.048 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'pbspro'
Jun-27 17:04:36.076 [main] DEBUG nextflow.script.ProcessConfig - Config settings `withLabel:process_medium` matches labels `process_medium` for process with name NFCORE_BACTMAP:BACTMAP:SUB_SAMPLING:MASH_SKETCH
Jun-27 17:04:36.078 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: pbspro
Jun-27 17:04:36.078 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'pbspro'
Jun-27 17:04:36.107 [main] DEBUG nextflow.script.ProcessConfig - Config settings `withLabel:process_low` matches labels `process_low` for process with name NFCORE_BACTMAP:BACTMAP:SUB_SAMPLING:RASUSA
Jun-27 17:04:36.108 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: pbspro
Jun-27 17:04:36.108 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'pbspro'
Jun-27 17:04:36.184 [main] DEBUG nextflow.script.ProcessConfig - Config settings `withLabel:process_medium` matches labels `process_medium` for process with name NFCORE_BACTMAP:BACTMAP:BWA_MEM
Jun-27 17:04:36.186 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: pbspro
Jun-27 17:04:36.188 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'pbspro'
Jun-27 17:04:36.205 [main] DEBUG nextflow.script.ProcessConfig - Config settings `withLabel:process_medium` matches labels `process_medium` for process with name NFCORE_BACTMAP:BACTMAP:BAM_SORT_SAMTOOLS:SAMTOOLS_SORT
Jun-27 17:04:36.205 [main] DEBUG nextflow.script.ProcessConfig - Config settings `withName:SAMTOOLS_SORT` matches process NFCORE_BACTMAP:BACTMAP:BAM_SORT_SAMTOOLS:SAMTOOLS_SORT
Jun-27 17:04:36.206 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: pbspro
Jun-27 17:04:36.206 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'pbspro'
Jun-27 17:04:36.214 [main] DEBUG nextflow.script.ProcessConfig - Config settings `withLabel:process_low` matches labels `process_low` for process with name NFCORE_BACTMAP:BACTMAP:BAM_SORT_SAMTOOLS:SAMTOOLS_INDEX
Jun-27 17:04:36.216 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: pbspro
Jun-27 17:04:36.216 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'pbspro'
Jun-27 17:04:36.232 [main] DEBUG nextflow.script.ProcessConfig - Config settings `withLabel:process_low` matches labels `process_low` for process with name NFCORE_BACTMAP:BACTMAP:BAM_SORT_SAMTOOLS:BAM_STATS_SAMTOOLS:SAMTOOLS_STATS
Jun-27 17:04:36.233 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: pbspro
Jun-27 17:04:36.234 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'pbspro'
Jun-27 17:04:36.250 [main] DEBUG nextflow.script.ProcessConfig - Config settings `withLabel:process_low` matches labels `process_low` for process with name NFCORE_BACTMAP:BACTMAP:BAM_SORT_SAMTOOLS:BAM_STATS_SAMTOOLS:SAMTOOLS_FLAGSTAT
Jun-27 17:04:36.250 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: pbspro
Jun-27 17:04:36.250 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'pbspro'
Jun-27 17:04:36.258 [main] DEBUG nextflow.script.ProcessConfig - Config settings `withLabel:process_low` matches labels `process_low` for process with name NFCORE_BACTMAP:BACTMAP:BAM_SORT_SAMTOOLS:BAM_STATS_SAMTOOLS:SAMTOOLS_IDXSTATS
Jun-27 17:04:36.261 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: pbspro
Jun-27 17:04:36.261 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'pbspro'
Jun-27 17:04:36.278 [main] DEBUG nextflow.script.ProcessConfig - Config settings `withLabel:process_medium` matches labels `process_medium` for process with name NFCORE_BACTMAP:BACTMAP:VARIANTS_BCFTOOLS:BCFTOOLS_MPILEUP
Jun-27 17:04:36.279 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: pbspro
Jun-27 17:04:36.279 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'pbspro'
Jun-27 17:04:36.285 [main] DEBUG nextflow.script.ProcessConfig - Config settings `withLabel:process_medium` matches labels `process_medium` for process with name NFCORE_BACTMAP:BACTMAP:VARIANTS_BCFTOOLS:BCFTOOLS_FILTER
Jun-27 17:04:36.285 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: pbspro
Jun-27 17:04:36.286 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'pbspro'
Jun-27 17:04:36.301 [main] DEBUG nextflow.script.ProcessConfig - Config settings `withLabel:process_low` matches labels `process_low` for process with name NFCORE_BACTMAP:BACTMAP:VCF2PSEUDOGENOME
Jun-27 17:04:36.303 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: pbspro
Jun-27 17:04:36.303 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'pbspro'
Jun-27 17:04:36.320 [main] DEBUG nextflow.script.ProcessConfig - Config settings `withLabel:process_low` matches labels `process_low` for process with name NFCORE_BACTMAP:BACTMAP:ALIGNPSEUDOGENOMES
Jun-27 17:04:36.323 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: pbspro
Jun-27 17:04:36.324 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'pbspro'
Jun-27 17:04:36.339 [main] DEBUG nextflow.script.ProcessConfig - Config settings `withLabel:process_medium` matches labels `process_medium` for process with name NFCORE_BACTMAP:BACTMAP:SNPSITES
Jun-27 17:04:36.341 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: pbspro
Jun-27 17:04:36.341 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'pbspro'
Jun-27 17:04:36.351 [main] DEBUG nextflow.script.ProcessConfig - Config settings `withLabel:process_medium` matches labels `process_medium` for process with name NFCORE_BACTMAP:BACTMAP:CREATE_PHYLOGENY:RAPIDNJ
Jun-27 17:04:36.352 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: pbspro
Jun-27 17:04:36.352 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'pbspro'
Jun-27 17:04:36.363 [main] DEBUG nextflow.script.ProcessConfig - Config settings `withLabel:process_medium` matches labels `process_medium` for process with name NFCORE_BACTMAP:BACTMAP:CREATE_PHYLOGENY:FASTTREE
Jun-27 17:04:36.363 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: pbspro
Jun-27 17:04:36.363 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'pbspro'
Jun-27 17:04:36.373 [main] DEBUG nextflow.script.ProcessConfig - Config settings `withLabel:process_medium` matches labels `process_medium` for process with name NFCORE_BACTMAP:BACTMAP:CREATE_PHYLOGENY:IQTREE
Jun-27 17:04:36.374 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: pbspro
Jun-27 17:04:36.374 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'pbspro'
Jun-27 17:04:36.379 [main] DEBUG nextflow.script.ProcessConfig - Config settings `withLabel:process_high` matches labels `process_high,process_long` for process with name NFCORE_BACTMAP:BACTMAP:CREATE_PHYLOGENY:RAXMLNG
Jun-27 17:04:36.379 [main] DEBUG nextflow.script.ProcessConfig - Config settings `withLabel:process_long` matches labels `process_high,process_long` for process with name NFCORE_BACTMAP:BACTMAP:CREATE_PHYLOGENY:RAXMLNG
Jun-27 17:04:36.379 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: pbspro
Jun-27 17:04:36.379 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'pbspro'
Jun-27 17:04:36.396 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: pbspro
Jun-27 17:04:36.396 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'pbspro'
Jun-27 17:04:36.469 [main] DEBUG nextflow.Session - Session aborted -- Cause: Access to 'FASTP.out' is undefined since the process 'FASTP' has not been invoked before accessing the output attribute
Jun-27 17:04:36.507 [main] DEBUG nextflow.Session - The following nodes are still active:
  [operator] splitCsv
  [operator] map
  [operator] map
  [operator] combine
  [operator] ifEmpty
  [operator] mix
  [operator] ifEmpty
  [operator] mix
  [operator] join
  [operator] ifEmpty
  [operator] mix
  [operator] ifEmpty
  [operator] mix
  [operator] map
  [operator] collect
  [operator] branch
  [operator] view
  [operator] map
  [operator] ifEmpty
  [operator] mix
  [operator] ifEmpty
  [operator] mix
  [operator] ifEmpty
  [operator] mix
  [operator] ifEmpty
  [operator] mix
  [operator] ifEmpty
  [operator] mix
  [operator] map
  [operator] collect
  [operator] collect
  [operator] ifEmpty
  [operator] collect
  [operator] collectFile

Jun-27 17:04:36.559 [main] ERROR nextflow.cli.Launcher - Access to 'FASTP.out' is undefined since the process 'FASTP' has not been invoked before accessing the output attribute
nextflow.exception.ScriptRuntimeException: Access to 'FASTP.out' is undefined since the process 'FASTP' has not been invoked before accessing the output attribute
        at nextflow.script.ProcessDef.getOut(ProcessDef.groovy:153)
        at jdk.internal.reflect.GeneratedMethodAccessor120.invoke(Unknown Source)
        at java.base/jdk.internal.reflect.DelegatingMethodAccessorImpl.invoke(DelegatingMethodAccessorImpl.java:43)
        at java.base/java.lang.reflect.Method.invoke(Method.java:566)
        at org.codehaus.groovy.reflection.CachedMethod.invoke(CachedMethod.java:107)
        at groovy.lang.MetaMethod.doMethodInvoke(MetaMethod.java:323)
        at org.codehaus.groovy.runtime.metaclass.MethodMetaProperty$GetBeanMethodMetaProperty.getProperty(MethodMetaProperty.java:76)
        at org.codehaus.groovy.runtime.callsite.GetEffectivePogoPropertySite.getProperty(GetEffectivePogoPropertySite.java:85)
        at org.codehaus.groovy.runtime.callsite.AbstractCallSite.callGetProperty(AbstractCallSite.java:329)
        at Script_c99ee09b$_runScript_closure1$_closure3.doCall(Script_c99ee09b:230)
        at java.base/jdk.internal.reflect.NativeMethodAccessorImpl.invoke0(Native Method)
        at java.base/jdk.internal.reflect.NativeMethodAccessorImpl.invoke(NativeMethodAccessorImpl.java:62)
        at java.base/jdk.internal.reflect.DelegatingMethodAccessorImpl.invoke(DelegatingMethodAccessorImpl.java:43)
        at java.base/java.lang.reflect.Method.invoke(Method.java:566)
        at org.codehaus.groovy.reflection.CachedMethod.invoke(CachedMethod.java:107)
        at groovy.lang.MetaMethod.doMethodInvoke(MetaMethod.java:323)
        at org.codehaus.groovy.runtime.metaclass.ClosureMetaClass.invokeMethod(ClosureMetaClass.java:274)
        at groovy.lang.MetaClassImpl.invokeMethod(MetaClassImpl.java:1035)
        at groovy.lang.Closure.call(Closure.java:412)
        at groovy.lang.Closure.call(Closure.java:406)
        at nextflow.script.WorkflowDef.run0(WorkflowDef.groovy:205)
        at nextflow.script.WorkflowDef.run(WorkflowDef.groovy:189)
        at nextflow.script.BindableDef.invoke_a(BindableDef.groovy:52)
        at nextflow.script.ComponentDef.invoke_o(ComponentDef.groovy:41)
        at nextflow.script.WorkflowBinding.invokeMethod(WorkflowBinding.groovy:94)
        at org.codehaus.groovy.runtime.metaclass.ClosureMetaClass.invokeOnDelegationObjects(ClosureMetaClass.java:408)
        at org.codehaus.groovy.runtime.metaclass.ClosureMetaClass.invokeMethod(ClosureMetaClass.java:350)
        at org.codehaus.groovy.runtime.callsite.PogoMetaClassSite.callCurrent(PogoMetaClassSite.java:61)
        at org.codehaus.groovy.runtime.callsite.CallSiteArray.defaultCallCurrent(CallSiteArray.java:51)
        at org.codehaus.groovy.runtime.callsite.AbstractCallSite.callCurrent(AbstractCallSite.java:171)
        at org.codehaus.groovy.runtime.callsite.AbstractCallSite.callCurrent(AbstractCallSite.java:176)
        at Script_14934cd2$_runScript_closure1$_closure3.doCall(Script_14934cd2:34)
        at java.base/jdk.internal.reflect.NativeMethodAccessorImpl.invoke0(Native Method)
        at java.base/jdk.internal.reflect.NativeMethodAccessorImpl.invoke(NativeMethodAccessorImpl.java:62)
        at java.base/jdk.internal.reflect.DelegatingMethodAccessorImpl.invoke(DelegatingMethodAccessorImpl.java:43)
        at java.base/java.lang.reflect.Method.invoke(Method.java:566)
        at org.codehaus.groovy.reflection.CachedMethod.invoke(CachedMethod.java:107)
        at groovy.lang.MetaMethod.doMethodInvoke(MetaMethod.java:323)
        at org.codehaus.groovy.runtime.metaclass.ClosureMetaClass.invokeMethod(ClosureMetaClass.java:274)
        at groovy.lang.MetaClassImpl.invokeMethod(MetaClassImpl.java:1035)
        at groovy.lang.Closure.call(Closure.java:412)
        at groovy.lang.Closure.call(Closure.java:406)
        at nextflow.script.WorkflowDef.run0(WorkflowDef.groovy:205)
        at nextflow.script.WorkflowDef.run(WorkflowDef.groovy:189)
        at nextflow.script.BindableDef.invoke_a(BindableDef.groovy:52)
        at nextflow.script.ComponentDef.invoke_o(ComponentDef.groovy:41)
        at nextflow.script.WorkflowBinding.invokeMethod(WorkflowBinding.groovy:94)
        at org.codehaus.groovy.runtime.metaclass.ClosureMetaClass.invokeOnDelegationObjects(ClosureMetaClass.java:408)
        at org.codehaus.groovy.runtime.metaclass.ClosureMetaClass.invokeMethod(ClosureMetaClass.java:350)
        at org.codehaus.groovy.runtime.callsite.PogoMetaClassSite.callCurrent(PogoMetaClassSite.java:61)
        at org.codehaus.groovy.runtime.callsite.CallSiteArray.defaultCallCurrent(CallSiteArray.java:51)
        at org.codehaus.groovy.runtime.callsite.AbstractCallSite.callCurrent(AbstractCallSite.java:171)
        at org.codehaus.groovy.runtime.callsite.AbstractCallSite.callCurrent(AbstractCallSite.java:176)
        at Script_14934cd2$_runScript_closure2$_closure4.doCall(Script_14934cd2:48)
        at java.base/jdk.internal.reflect.NativeMethodAccessorImpl.invoke0(Native Method)
        at java.base/jdk.internal.reflect.NativeMethodAccessorImpl.invoke(NativeMethodAccessorImpl.java:62)
        at java.base/jdk.internal.reflect.DelegatingMethodAccessorImpl.invoke(DelegatingMethodAccessorImpl.java:43)
        at java.base/java.lang.reflect.Method.invoke(Method.java:566)
        at org.codehaus.groovy.reflection.CachedMethod.invoke(CachedMethod.java:107)
        at groovy.lang.MetaMethod.doMethodInvoke(MetaMethod.java:323)
        at org.codehaus.groovy.runtime.metaclass.ClosureMetaClass.invokeMethod(ClosureMetaClass.java:274)
        at groovy.lang.MetaClassImpl.invokeMethod(MetaClassImpl.java:1035)
        at groovy.lang.Closure.call(Closure.java:412)
        at groovy.lang.Closure.call(Closure.java:406)
        at nextflow.script.WorkflowDef.run0(WorkflowDef.groovy:205)
        at nextflow.script.WorkflowDef.run(WorkflowDef.groovy:189)
        at nextflow.script.BindableDef.invoke_a(BindableDef.groovy:52)
        at nextflow.script.ChainableDef$invoke_a.call(Unknown Source)
        at org.codehaus.groovy.runtime.callsite.CallSiteArray.defaultCall(CallSiteArray.java:47)
        at org.codehaus.groovy.runtime.callsite.AbstractCallSite.call(AbstractCallSite.java:125)
        at org.codehaus.groovy.runtime.callsite.AbstractCallSite.call(AbstractCallSite.java:139)
        at nextflow.script.BaseScript.runDsl2(BaseScript.groovy:208)
        at nextflow.script.BaseScript.run(BaseScript.groovy:217)
        at nextflow.script.ScriptParser.runScript(ScriptParser.groovy:220)
        at nextflow.script.ScriptRunner.run(ScriptRunner.groovy:212)
        at nextflow.script.ScriptRunner.execute(ScriptRunner.groovy:120)
        at nextflow.cli.CmdRun.run(CmdRun.groovy:337)
        at nextflow.cli.Launcher.run(Launcher.groovy:480)
        at nextflow.cli.Launcher.main(Launcher.groovy:639)
Jun-27 17:04:36.601 [Actor Thread 6] DEBUG nextflow.file.SortFileCollector - FileCollector temp dir not removed: null

System

Hardware: HPC
Executor: PBSPRO
OS: SUSE
Version 12

Nextflow Installation

Version: 22.04.4

Container engine

Engine: singularity
version: 3.9.8-1

BACTMAP: Convert all param docs to JSON schema

Hi!

this is not necessarily an issue with the pipeline, but in order to streamline the documentation group next week for the hackathon, I'm opening issues in all repositories / pipeline repos that might need this update to switch from parameter docs to auto-generated documentation based on the JSON schema.

This will then supersede any further parameter documentation, thus making things a bit easier :-)

If this doesn't apply (anymore), please close the issue. Otherwise, I'm hoping to have some helping hands on this next week in the documentation team on Slack https://nfcore.slack.com/archives/C01QPMKBYNR

Memory error

Hi,
Thanks for devloping this nice SNP pipeline, .
I have used bactmap on ~200 samples using conda . bactmap crashed however every time with an error related to the memory load on the system. I used with the following setting in the command line --max_memory 1.GB --max_cpus but didn't help.

This was my full command
nextflow run nf-core/bactmap --input samplesSheet_01Feb2022_160557.tsv --reference <ref> -profile conda --trim --outdir results_bactmap -resume --raxmlng --genome_size 1.9m --max_memory 1.GB --max_cpus 2
I am working on a Centos machine with 1008G ram and 112 cpus.

I appreciate your help. Thank you,

Missing TEMPLATE branch

Hi,
I'm looking at all pipelines, and I noticed that this one is on branch.
All pipelines should have a TEMPLATE branch.

Incorporate nf-core modules into bactmap

[ ] rasusa
[ ] mash/sketch
[ ] gubbins
[x] snp-sites
[x] fasttree
[ ] iqtree
[ ] raxml-ng

snp-sites DSL2 module

Option to downsample before fastp

In my most recent analysis, each of the fastq files for a sample was 8GB (yes I know) which led fastp to give up the ghost, I think because of memory but there wasn't an obvious error message. I'm not sure there's much we can do about this as we subsample after fastp. I guess it may be a pretty rare use case but I thought it worth mentioning

Add documentation

Update README.md, output.md, usage.md

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