Usage

Table of contents

• Table of contents
• Running the pipeline
  • Quick Start
  • Updating the pipeline
  • Reproducibility
  • Automatic Resubmission
• Core Nextflow arguments
  • -profile
  • -resume
  • -c
  • Running in the background
• Pipeline Options
  • Input
  • Input Data Additional Options
  • Reference Genomes
  • Output
  • Other run specific parameters
  • Step skipping parameters
  • Complexity Filtering Options
  • Adapter Clipping and Merging Options
  • Read Mapping Parameters
  • Removal of Host-Mapped Reads
  • Read Filtering and Conversion Parameters
  • Read DeDuplication Parameters
  • Library Complexity Estimation Parameters
  • DNA Damage Assessment Parameters
  • Feature Annotation Statistics
  • BAM Trimming Parameters
  • Genotyping Parameters
  • Consensus Sequence Generation
  • SNP Table Generation
  • Mitochondrial to Nuclear Ratio
  • Human Sex Determination
  • Human Nuclear Contamination
  • Metagenomic Screening
  • Metagenomic Authentication
  • Clean up
• Troubleshooting and FAQs
  • My pipeline update doesn’t seem to do anything
  • Input files not found
  • I am only getting output for a single sample although I specified multiple with wildcards
  • The pipeline crashes almost immediately with an early pipeline step
  • The pipeline has crashed with an error but Nextflow is still running
  • I get a exceeded job memory limit error
  • I get a file name collision error during merging
  • I specified a module and it didn’t produce the expected output
  • I get a unable to acquire lock
• Tutorials
  • Tutorial - How to investigate a failed run
  • Tutorial - What are Profiles and How To Use Them
  • Tutorial - How to set up nf-core/eager for human population genetics
  • Tutorial - How to set up nf-core/eager for metagenomic screening
  • Tutorial - How to set up nf-core/eager for pathogen genomics

Running the pipeline

Quick Start

Before you start you should change into the output directory you wish your results to go in. This will guarantee, that when you start the Nextflow job, it will place all the log files and ‘working’ folders in the corresponding output directory, (and not wherever else you may have executed the run from)

The typical command for running the pipeline is as follows:

nextflow run nf-core/eager --input '*_R{1,2}.fastq.gz' --fasta 'some.fasta' -profile standard,docker

where the reads are from FASTQ files of the same pairing.

This will launch the pipeline with the docker configuration profile. See below for more information about profiles.

Note that the pipeline will create the following files in your working directory:

work            # Directory containing the Nextflow working files
results         # Finished results (configurable, see below)
.nextflow.log   # Log file from Nextflow
                # Other Nextflow hidden files, eg. history of pipeline runs and old logs.

To see the the nf-core/eager pipeline help message run: nextflow run nf-core/eager --help

If you want to configure your pipeline interactively using a graphical user interface, please visit nf-co.re launch. Select the eager pipeline and the version you intend to run, and follow the on-screen instructions to create a config for your pipeline run.

Updating the pipeline

When you run the above command, Nextflow automatically pulls the pipeline code from GitHub and stores it as a cached version. When running the pipeline after this, it will always use the cached version if available - even if the pipeline has been updated since. To make sure that you’re running the latest version of the pipeline, make sure that you regularly update the cached version of the pipeline:

nextflow pull nf-core/eager

Reproducibility

It’s a good idea to specify a pipeline version when running the pipeline on your data. This ensures that a specific version of the pipeline code and software are used when you run your pipeline. If you keep using the same tag, you’ll be running the same version of the pipeline, even if there have been changes to the code since.

First, go to the nf-core/eager releases page and find the latest version number - numeric only (eg. 2.2.0). Then specify this when running the pipeline with -r (one hyphen) - eg. -r 2.2.0.

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.

Additionally, nf-core/eager pipeline releases are named after Swabian German Cities. The first release V2.0 is named “Kaufbeuren”. Future releases are named after cities named in the Swabian league of Cities.

Automatic Resubmission

By default, if a pipeline step fails, nf-core/eager will resubmit the job with twice the amount of CPU and memory. This will occur two times before failing.

Core Nextflow arguments

NB: These options are part of Nextflow and use a single hyphen (pipeline parameters use a double-hyphen).

-profile

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, 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.

The pipeline also dynamically loads configurations from https://github.com/nf-core/configs when it runs, making multiple config profiles for various institutional clusters available at run time. For more information and to see if your system is available in these configs please see the nf-core/configs documentation.

Note that multiple profiles can be loaded, for example: -profile test,docker - the order of arguments is important! They are loaded in sequence, so later profiles can overwrite earlier profiles.

If -profile is not specified, the pipeline will run locally and expect all software to be installed and available on the PATH. This is not recommended.

Important: If running nf-core/eager on a cluster - ask your system administrator what profile to use.

• docker
  • A generic configuration profile to be used with Docker
  • Pulls software from Docker Hub: nfcore/eager
• singularity
  • A generic configuration profile to be used with Singularity
  • Pulls software from Docker Hub: nfcore/eager
• conda
  • Please only use Conda as a last resort i.e. when it’s not possible to run the pipeline with Docker or Singularity.
  • A generic configuration profile to be used with Conda
  • Pulls most software from Bioconda
• test
  • A profile with a complete configuration for automated testing
  • Includes links to test data so needs no other parameters

Institution Specific Profiles These are profiles specific to certain HPC clusters, and are centrally maintained at nf-core/configs. Those listed below are regular users of nf-core/eager, if you don’t see your own institution here check the nf-core/configs repository.

• uzh
  • A profile for the University of Zurich Research Cloud
  • Loads Singularity and defines appropriate resources for running the pipeline.
• binac
  • A profile for the BinAC cluster at the University of Tuebingen 0 Loads Singularity and defines appropriate resources for running the pipeline
• shh
  • A profile for the S/CDAG cluster at the Department of Archaeogenetics of the Max Planck Institute for the Science of Human History
  • Loads Singularity and defines appropriate resources for running the pipeline

Pipeline Specific Institution Profiles There are also pipeline-specific institution profiles. I.e., we can also offer a profile which sets special resource settings to specific steps of the pipeline, which may not apply to all pipelines. This can be seen at nf-core/configs under conf/pipelines/eager/.

We currently offer a nf-core/eager specific profile for

• shh
  • A profiler for the S/CDAG cluster at the Department of Archaeogenetics of the Max Planck Institute for the Science of Human History
  • In addition to the nf-core wide profile, this also sets the MALT resources to match our commonly used databases

Further institutions can be added at nf-core/configs. Please ask the eager developers to add your institution to the list above, if you add one!

-resume

Specify this when restarting a pipeline. Nextflow will used cached results from any pipeline steps where the inputs are the same, continuing from where it got to previously.

You can also supply a run name to resume a specific run: -resume [run-name]. Use the nextflow log command to show previous run names.

-c

Specify the path to a specific config file (this is a core Nextflow command). See the nf-core website documentation for more information.

Custom resource 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 an error code of 143 (exceeded requested resources) it will automatically resubmit with higher requests (2 x original, then 3 x original). If it still fails after three times then the pipeline is stopped.

Whilst these default requirements will hopefully work for most people with most data, you may find that you want to customise the compute resources that the pipeline requests. You can do this by creating a custom config file. For example, to give the workflow process star 32GB of memory, you could use the following config:

process {
  withName: bwa {
    memory = 32.GB
  }
}

See the main Nextflow documentation for more information.

If you are likely to be running nf-core pipelines regularly it may be a good idea to request that your custom config file is uploaded to the nf-core/configs git repository. Before you do this please can you test that the config file works with your pipeline of choice using the -c parameter (see definition below). You can then create a pull request to the nf-core/configs repository with the addition of your config file, associated documentation file (see examples in nf-core/configs/docs), and amending nfcore_custom.config to include your custom profile.

If you have any questions or issues please send us a message on Slack on the #configs channel.

-name

Name for the pipeline run. If not specified, Nextflow will automatically generate a random mnemonic.

This is used in the MultiQC report (if not default) and in the summary HTML / e-mail (always).

NB: Single hyphen (core Nextflow option)

Running in the background

Nextflow handles job submissions and supervises the running jobs. The Nextflow process must run until the pipeline is finished.

Nextflow handles job submissions on SLURM or other environments, and supervises running the jobs. Thus the Nextflow process must run until the pipeline is finished. We recommend that you put the process running in the background through screen / tmux or similar tool. Alternatively you can run Nextflow within a cluster job submitted your job scheduler.

To create a screen session:

screen -R nf-core/eager

To disconnect, press ctrl+a then d.

To reconnect, type:

screen -r nf-core/eager

to end the screen session while in it type exit.

Alternatively, the Nextflow -bg flag launches Nextflow in the background, detached from your terminal so that the workflow does not stop if you log out of your session. The logs are saved to a file.

Nextflow memory requirements

In some cases, the Nextflow Java virtual machines can start to request a large amount of memory. We recommend adding the following line to your environment to limit this (typically in ~/.bashrc or ~./bash_profile):

NXF_OPTS='-Xms1g -Xmx4g'

Pipeline Options

Input

--input

There are two possible ways of supplying input sequencing data to nf-core/eager. The most efficient but more simplistic is supplying direct paths (with wildcards) to your FASTQ or BAM files, with each file or pair being considered a single library and each one run independently. TSV input requires creation of an extra file by the user and extra metadata, but allows more powerful lane and library merging.

Direct Input Method

This method is where you specify with --input, the path locations of FASTQ (optionally gzipped) or BAM file(s). This option is mutually exclusive to the TSV input method, which is used for more complex input configurations such as lane and library merging.

When using the direct method of --input you can specify one or multiple samples in one or more directories files. File names must be unique, even if in different directories.

By default, the pipeline assumes you have paired-end data. If you want to run single-end data you must specify --single_end

For example, for a single set of FASTQs, or multiple paired-end FASTQ files in one directory, you can specify:

--input 'path/to/data/sample_*_{1,2}.fastq.gz'

If you have multiple files in different directories, you can use additional wildcards (*) e.g.:

--input 'path/to/data/*/sample_*_{1,2}.fastq.gz'

⚠️ It is not possible to run a mixture of single-end and paired-end files in one run with the paths --input method! Please see the TSV input method for possibilities.

Please note the following requirements:

1. Valid file extensions: .fastq.gz, .fastq, .fq.gz, .fq, .bam.
2. The path must be enclosed in quotes
3. The path must have at least one * wildcard character
4. When using the pipeline with paired end data, the path must use {1,2} notation to specify read pairs.
5. Files names must be unique, having files with the same name, but in different directories is not sufficient
   • This can happen when a library has been sequenced across two sequencers on the same lane. Either rename the file, try a symlink with a unique name, or merge the two FASTQ files prior input.
6. Due to limitations of downstream tools (e.g. FastQC), sample IDs may be truncated after the first . in the name, Ensure file names are unique prior to this!
7. For input BAM files you should provide a small decoy reference genome with pre-made indices, e.g. the human mtDNA or phiX genome, for the mandatory parameter --fasta in order to avoid long computational time for generating the index files of the reference genome, even if you do not actual need a reference genome for any downstream analyses.

TSV Input Method

Alternatively to the direct input method, you can supply to --input a path to a TSV file that contains paths to FASTQ/BAM files and additional metadata. This allows for more complex procedures such as merging of sequencing data across lanes, sequencing runs, sequencing configuration types, and samples.

The use of the TSV --input method is recommended when performing more complex procedures such as lane or library merging. You do not need to specify --single_end, --bam, --colour_chemistry, -udg_type etc. when using TSV input - this is defined within the TSV file itself. You can only supply a single TSV per run (i.e. --input '*.tsv' will not work).

This TSV should look like the following:

A template can be taken from here.

⚠️ Cells must not contain spaces before or after strings, as this will make the TSV unreadable by Nextflow. Strings containing spaces should be wrapped in quotes.

When using TSV_input, nf-core/eager will merge FASTQ files of libraries with the same Library_ID but different Lanes values after adapter clipping (and merging), assuming all other metadata columns are the same. If you have the same Library_ID but with different SeqType, this will be merged directly after mapping prior BAM filtering. Finally, it will also merge BAM files with the same Sample_ID but different Library_ID after duplicate removal, but prior to genotyping. Please see caveats to this below.

Column descriptions are as follows:

• Sample_Name: A text string containing the name of a given sample of which there can be multiple libraries. All libraries with the same sample name and same SeqType will be merged after deduplication.
• Library_ID: A text string containing a given library, which there can be multiple sequencing lanes (with the same SeqType).
• Lane: A number indicating which lane the library was sequenced on. Files from the libraries sequenced on different lanes (and different SeqType) will be concatenated after read clipping and merging.
• Colour Chemistry A number indicating whether the Illumina sequencer the library was sequenced on was a 2 (e.g. Next/NovaSeq) or 4 (Hi/MiSeq) colour chemistry machine. This informs whether poly-G trimming (if turned on) should be performed.
• SeqType: A text string of either ‘PE’ or ‘SE’, specifying paired end (with both an R1 [or forward] and R2 [or reverse]) and single end data (only R1 [forward], or BAM). This will affect lane merging if different per library.
• Organism: A text string of the organism name of the sample or ‘NA’. This currently has no functionality and can be set to ‘NA’, but will affect lane/library merging if different per library
• Strandedness: A text string indicating whether the library type is ‘single’ or ‘double’. This will affect lane/library merging if different per library.
• UDG_Treatment: A text string indicating whether the library was generated with UDG treatment - either ‘full’, ‘half’ or ‘none’. Will affect lane/library merging if different per library.
• R1: A text string of a file path pointing to a forward or R1 FASTQ file. This can be used with the R2 column. File names must be unique, even if they are in different directories.
• R2: A text string of a file path pointing to a reverse or R2 FASTQ file, or ‘NA’ when single end data. This can be used with the R1 column. File names must be unique, even if they are in different directories.
• BAM: A text string of a file path pointing to a BAM file, or ‘NA’. Cannot be specified at the same time as R1 or R2, both of which should be set to ‘NA’

For example, the following TSV table:

will have the following effects:

• After AdapterRemoval, and prior to mapping, FASTQ files from lane 7 and lane 8 with the same SeqType (and all other metadata columns) will be concatenated together for each Library.
• After mapping, and prior BAM filtering, BAM files with the same with different SeqType (but with all other metadata columns the same) will be merged together for each Library.
• After duplicate removal, BAM files with Library_IDs with the same Sample_Name and the same UDG_Treatment will be merged together.
• If BAM trimming is turned on, all post-trimming BAMs (i.e. non-UDG and half-UDG ) will be merged with UDG-treated (untreated) BAMs, if they have the same Sample_Name.

Note the following important points and limitations for setting up:

• The TSV must use actual tabs (not spaces) between cells.
• File names must be unique regardless of file path, due to risk of over-writing (see: https://github.com/nextflow-io/nextflow/issues/470).
  • If it is ‘too late’ and you already have duplicate file names, a workaround is to concatenate the FASTQ files together and supply this to a nf-core/eager run. The only downside is that you will not get independent FASTQC results for each file.
• Lane IDs must be unique for each sequencing of each library.
  • If you have a library sequenced e.g. on Lane 8 of two HiSeq runs, you can give a fake lane ID (e.g. 20) for one of the FASTQs, and the libraries will still be processed correctly.
  • This also applies to the SeqType column, i.e. with the example above, if one run is PE and one run is SE, you need to give fake lane IDs to one of the runs as well.
• All BAM files must be specified as SE under SeqType.
  • You should provide a small decoy reference genome with pre-made indices, e.g. the human mtDNA or phiX genome, for the mandatory parameter --fasta in order to avoid long computational time for generating the index files of the reference genome, even if you do not actual need a reference genome for any downstream analyses.
• nf-core/eager will only merge multiple lanes of sequencing runs with the same single-end or paired-end configuration
• Accordingly nf-core/eager will not merge lanes of FASTQs with BAM files (unless you use --run_convertbam), as only FASTQ files are lane-merged together.
• Same libraries that are sequenced on different sequencing configurations (i.e single- and paired-end data), will be merged after mapping and will always be considered ‘paired-end’ during downstream processes
  • Important running DeDup in this context is not recommended, as PE and SE data at the same position will not be evaluated as duplicates. Therefore not all duplicates will be removed.
  • When you wish to run PE/SE data together -dedupper markduplicates is therefore preferred.
  • An error will be thrown if you try to merge both PE and SE and also supply --skip_merging.
  • If you truly want to mix SE data and PE data but using mate-pair info for PE mapping, please run FASTQ preprocessing mapping manually and supply BAM files for downstream processing by nf-core/eager
  • If you regularly want to run the situation above, please leave a feature request on github.
• DamageProfiler, NuclearContamination, MTtoNucRatio and PreSeq are performed on each unique library separately after deduplication (but prior same-treated library merging).
• nf-core/eager functionality such as --run_trim_bam will be applied to only non-UDG (UDG_Treatment: none) or half-UDG (UDG_Treatment: half) libraries.
• Qualimap is run on each sample, after merging of libraries (i.e. your values will reflect the values of all libraries combined - after being damage trimmed etc.).
• Genotyping will be typically performed on each sample independently, as normally all libraries will have been merged together. However, if you have a mixture of single-stranded and double-stranded libraries, you will normally need to genotype separately. In this case you must give each the SS and DS libraries distinct Sample_IDs; otherwise you will receive a file collision error in steps such as sexdeterrmine, and then you will need to merge these yourself. We will consider changing this behaviour in the future if there is enough interest.

--udg_type

Defines whether Uracil-DNA glycosylase (UDG) treatment was used to remove DNA damage on the sequencing libraries.

Specify 'none' if no treatment was performed. If you have partial UDG treated data (Rohland et al 2016), specify 'half'. If you have complete UDG treated data (Briggs et al. 2010), specify 'full'.

When also using PMDtools specifying 'half' will use a different model for DNA damage assessment in PMDTools (PMDtools: --UDGhalf). Specify 'full' and the PMDtools DNA damage assessment will use CpG context only (PMDtools: --CpG). Default: 'none'.

Tip: You should provide a small decoy reference genome with pre-made indices, e.g. the human mtDNA genome, for the mandatory parameter --fasta in order to avoid long computational time for generating the index files of the reference genome, even if you do not actual need a reference genome for any downstream analyses.

--single_stranded

Indicates libraries are single stranded.

Currently only affects MALTExtract, where it will switch on damage patterns calculation mode to single-stranded, (MaltExtract: --singleStranded) and genotyping with pileupCaller where a different method is used (pileupCaller: --singleStrandMode). Default: false.

Only required when using the ‘Path’ method of --input.

--single_end

Indicates libraries were sequenced with single-end sequencing chemistries (i.e. only a R1 file is present). If not supplied, input data assumed to be paired-end by default.

Only required when using the ‘Path’ method of --input.

--colour_chemistry

Specifies which Illumina colour chemistry a library was sequenced with. This informs whether to perform poly-G trimming (if --complexity_filter_poly_g is also supplied). Only 2 colour chemistry sequencers (e.g. NextSeq or NovaSeq) can generate uncertain poly-G tails (due to ‘G’ being indicated via a no-colour detection). Default is ‘4’ to indicate e.g. HiSeq or MiSeq platforms, which do not require poly-G trimming. Options: 2, 4. Default: 4

Only required when using the ‘Path’ method of --input.

--bam

Specifies the input file type to --input is in BAM format. This will automatically also apply --single_end.

Only required when using the ‘Path’ method of --input.

Input Data Additional Options

--snpcapture_bed

Can be used to set a path to a BED file (3/6 column format) of SNP positions of a reference genome, to calculate SNP captured libraries on-target efficiency. This should be used for array or in-solution SNP capture protocols such as 390K, 1240K, etc. If supplied, on-target metrics are automatically generated for you by qualimap.

--run_convertinputbam

Allows you to convert an input BAM file back to FASTQ for downstream processing. Note this is required if you need to perform AdapterRemoval and/or polyG clipping.

If not turned on, BAMs will automatically be sent to post-mapping steps.

Reference Genomes

All nf-core/eager runs require a reference genome in FASTA format to map reads against to.

In addition we provide various options for indexing of different types of reference genomes (based on the tools used in the pipeline). nf-core/eager can index reference genomes for you (with options to save these for other analysis), but you can also supply your pre-made indices.

Supplying pre-made indices saves time in pipeline execution and is especially advised when running multiple times on the same cluster system, for example. You can even add a resource specific profile that sets paths to pre-computed reference genomes, saving time when specifying these.

⚠️ you must always supply a reference file. If you want to use functionality that does not require one, supply a small decoy genome such as phiX or the human mtDNA genome.

--fasta

You specify the full path to your reference genome here. The FASTA file can have any file suffix, such as .fasta, .fna, .fa, .FastA etc. You may also supply a gzipped reference files, which will be unzipped automatically for you.

For example:

--fasta '/<path>/<to>/my_reference.fasta'

You need to provide an input FASTA even if you do not do any mapping (e.g. supplying BAM files). You should use a small decoy reference genome with pre-made indices, e.g. the human mtDNA genome, for the mandatory parameter --fasta in order to avoid long computational time for generating the index files of the reference genome.

If you don’t specify appropriate --bwa_index, --fasta_index parameters (see below), the pipeline will create these indices for you automatically. Note that you can save the indices created for you for later by giving the --save_reference flag. You must select either a --fasta or --genome

--genome (using iGenomes)

Alternatively, the pipeline config files come bundled with paths to the Illumina iGenomes reference index files. If running with Docker or AWS, the configuration is set up to use the AWS-iGenomes resource.

There are 31 different species supported in the iGenomes references. To run the pipeline, you must specify which to use with the --genome flag.

You can find the keys to specify the genomes in the iGenomes config file under conf/ on the nf-core/eager GitHub repository. Common genomes that are supported are:

• Human
  • --genome GRCh37
  • --genome GRCh38
• Mouse *
  • --genome GRCm38
• Drosophila *
  • --genome BDGP6
• S. cerevisiae *
  • --genome 'R64-1-1'

* Not bundled with nf-core eager by default.

Note that you can use the same configuration set-up to save sets of reference files for your own use, even if they are not part of the iGenomes resource. See the Nextflow documentation for instructions on where to save such a file.

The syntax for this reference configuration is as follows:

params {
  genomes {
    'GRCh37' {
      fasta   = '<path to the iGenomes genome fasta file>'
    }
    // Any number of additional genomes, key is used with --genome
  }
}

You must select either a --fasta or --genome

--bwa_index

If you want to use pre-existing bwa index indices, please supply the directory to the FASTA you also specified in --fasta (see above). nf-core/eager will automagically detect the index files by searching for the FASTA filename with the corresponding bwa index file suffixes.

For example:

nextflow run nf-core/eager \
-profile test,docker \
--input '*{R1,R2}*.fq.gz'
--fasta 'results/reference_genome/bwa_index/BWAIndex/Mammoth_MT_Krause.fasta' \
--bwa_index 'results/reference_genome/bwa_index/BWAIndex/'

bwa index does not give you an option to supply alternative suffixes/names for these indices. Thus, the file names generated by this command must not be changed, otherwise nf-core/eager will not be able to find them.

--bt2_index

If you want to use pre-existing bt2 index indices, please supply the directory to the FASTA you also specified in --fasta (see above). nf-core/eager will automagically detect the index files by searching for the FASTA filename with the corresponding bt2 index file suffixes.

For example:

nextflow run nf-core/eager \
-profile test,docker \
--input '*{R1,R2}*.fq.gz'
--fasta 'results/reference_genome/bwa_index/BWAIndex/Mammoth_MT_Krause.fasta' \
--bwa_index 'results/reference_genome/bt2_index/BT2Index/'

bowtie2-build does not give you an option to supply alternative suffixes/names for these indices. Thus, the file names generated by this command must not be changed, otherwise nf-core/eager will not be able to find them.

--fasta_index

If you want to use a pre-existing samtools faidx index, use this to specify the required FASTA index file for the selected reference genome. This should be generated by samtools faidx and has a file suffix of .fai

For example:

--fasta_index 'Mammoth_MT_Krause.fasta.fai'

--seq_dict

If you want to use a pre-existing picard CreateSequenceDictionary dictionary file, use this to specify the required .dict file for the selected reference genome.

For example:

--seq_dict 'Mammoth_MT_Krause.dict'

--large_ref

This parameter is required to be set for large reference genomes. If your reference genome is larger than 3.5GB, the samtools index calls in the pipeline need to generate CSI indices instead of BAI indices to compensate for the size of the reference genome (with samtools: -c). This parameter is not required for smaller references (including the human hg19 or grch37/grch38 references), but >4GB genomes have been shown to need CSI indices. Default: off

modifies SAMtools index command: -c

--save_reference

Use this if you do not have pre-made reference FASTA indices for bwa, samtools and picard. If you turn this on, the indices nf-core/eager generates for you and will be saved in the <your_output_dir>/results/reference_genomes for you. If not supplied, nf-core/eager generated index references will be deleted.

Output

--outdir

The output directory where the results will be saved.

-w / -work-dir

The output directory where intermediate files will be saved. It is highly recommended that this is the same path as --outdir, otherwise you may ‘lose’ your intermediate files if you need to re-run a pipeline. By default, if this flag is not given, the intermediate files will be saved in a work/ and .nextflow/ directory from wherever you have run nf-core/eager from.

--publish_dir_mode

Nextflow mode for ‘publishing’ final results files i.e. how to move final files into your --outdir from working directories. Options: ‘symlink’, ‘rellink’, ‘link’, ‘copy’, ‘copyNoFollow’, ‘move’. Default: ‘copy’.

It is recommended to select copy (default) if you plan to regularly delete intermediate files from work/.

Other run specific parameters

--max_memory

Use to set a top-limit for the default memory requirement for each process. Should be a string in the format integer-unit. eg. --max_memory '8.GB'. If not specified, will be taken from the configuration in the -profile flag.

--max_time

Use to set a top-limit for the default time requirement for each process. Should be a string in the format integer-unit. eg. --max_time '2.h'. If not specified, will be taken from the configuration in the -profile flag.

--max_cpus

When not using a institute specific -profile, you can use this parameter to set a top-limit for the default CPU requirement for each process. This is not the maximum number of CPUs that can be used for the whole pipeline, but the maximum number of CPUs each program can use for each program submission (known as a process).

Do not set this higher than what is available on your workstation or computing node can provide. If you’re unsure, ask your local IT administrator for details on compute node capabilities! Should be a string in the format integer-unit. eg. --max_cpus 1. If not specified, will be taken from the configuration in the -profile flag.

--email

Set this parameter to your e-mail address to get a summary e-mail with details of the run sent to you when the workflow exits. If set in your user config file (~/.nextflow/config) then you don’t need to specify this on the command line for every run.

Note that this functionality requires either mail or sendmail to be installed on your system.

--email_on_fail

Set this parameter to your e-mail address to get a summary e-mail with details of the run if it fails. Normally would be the same as in --email. If set in your user config file (~/.nextflow/config) then you don’t need to specify this on the command line for every run.

Note that this functionality requires either mail or sendmail to be installed on your system.

--plaintext_email

Set to receive plain-text e-mails instead of HTML formatted.

--monochrome_logs

Set to disable colourful command line output and live life in monochrome.

--multiqc_config

Specify a path to a custom MultiQC configuration file.

--custom_config_version

Provide git commit id for custom Institutional configs hosted at nf-core/configs. This was implemented for reproducibility purposes. Default is set to master.

\#\# Download and use config file with following git commit id
--custom_config_version d52db660777c4bf36546ddb188ec530c3ada1b96

Step skipping parameters

Some of the steps in the pipeline can be executed optionally. If you specify specific steps to be skipped, there won’t be any output related to these modules.

--skip_fastqc

Turns off FastQC pre- and post-Adapter Removal, to speed up the pipeline. Use of this flag is most common when data has been previously pre-processed and the post-Adapter Removal mapped reads are being re-mapped to a new reference genome.

--skip_adapterremoval

Turns off adapter trimming and paired-end read merging. Equivalent to setting both --skip_collapse and --skip_trim.

--skip_preseq

Turns off the computation of library complexity estimation.

--skip_deduplication

Turns off duplicate removal methods DeDup and MarkDuplicates respectively. No duplicates will be removed on any data in the pipeline.

--skip_damage_calculation

Turns off the DamageProfiler module to compute DNA damage profiles.

--skip_qualimap

Turns off QualiMap and thus does not compute coverage and other mapping metrics.

Complexity Filtering Options

More details can be seen in the fastp documentation

If using TSV input, this is performed per lane separately.

--complexity_filter_poly_g

Performs a poly-G tail removal step in the beginning of the pipeline using fastp, if turned on. This can be useful for trimming ploy-G tails from short-fragments sequenced on two-colour Illumina chemistry such as NextSeqs (where no-fluorescence is read as a G on two-colour chemistry), which can inflate reported GC content values.

--complexity_filter_poly_g_min

This option can be used to define the minimum length of a poly-G tail to begin low complexity trimming. By default, this is set to a value of 10 unless the user has chosen something specifically using this option.

Modifies fastp parameter: --poly_g_min_len

Adapter Clipping and Merging Options

These options handle various parts of adapter clipping and read merging steps.

More details can be seen in the AdapterRemoval documentation

If using TSV input, this is performed per lane separately.

⚠️ --skip_trim will skip adapter clipping AND quality trimming (n, base quality). It is currently not possible skip one or the other.

--clip_forward_adaptor

Defines the adapter sequence to be used for the forward read. By default, this is set to 'AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC'.

Modifies AdapterRemoval parameter: --adapter1

--clip_reverse_adaptor

Defines the adapter sequence to be used for the reverse read in paired end sequencing projects. This is set to 'AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTA' by default.

Modifies AdapterRemoval parameter: --adapter2

--clip_readlength

Defines the minimum read length that is required for reads after merging to be considered for downstream analysis after read merging. Default is 30.

Note that performing read length filtering at this step is not reliable for correct endogenous DNA calculation, when you have a large percentage of very short reads in your library - such as retrieved in single-stranded library protocols. When you have very few reads passing this length filter, it will artificially inflate your endogenous DNA by creating a very small denominator. In these cases it is recommended to set this to 0, and use --bam_filter_minreadlength instead, to filter out ‘un-usable’ short reads after mapping.

Modifies AdapterRemoval parameter: --minlength

--clip_min_read_quality

Defines the minimum read quality per base that is required for a base to be kept. Individual bases at the ends of reads falling below this threshold will be clipped off. Default is set to 20.

Modifies AdapterRemoval parameter: --minquality

--clip_min_adap_overlap

Sets the minimum overlap between two reads when read merging is performed. Default is set to 1 base overlap.

Modifies AdapterRemoval parameter: --minadapteroverlap

--skip_collapse

Turns off the paired-end read merging.

For example

--skip_collapse  --input '*_{R1,R2}_*.fastq'

It is important to use the paired-end wildcard globbing, as --skip_collapse can only be used on paired-end data!

⚠️ If you provide this option together with --clip_readlength set to something (as is by default), you may end up removing single reads from either the pair1 or pair2 file. These will be NOT be mapped when aligning with either bwa or bowtie, as both can only accept one (forward) or two (forward and reverse) FASTQs as input.

Modifies AdapterRemoval parameter: --collapse

--skip_trim

Turns off adapter AND quality trimming.

For example:

--skip_trim  --input '*.fastq'

⚠️ it is not possible to keep quality trimming (n or base quality) on, and skip adapter trimming.

⚠️ it is not possible to turn off one or the other of quality trimming or n trimming. i.e. —trimns —trimqualities are both given or neither. However setting quality in --clip_min_read_quality to 0 would theoretically turn off base quality trimming.

Modifies AdapterRemoval parameters: --trimns --trimqualities --adapter1 --adapter2

--preserve5p

Turns off quality based trimming at the 5p end of reads when any of the —trimns, —trimqualities, or —trimwindows options are used. Only 3p end of reads will be removed.

This also entirely disables quality based trimming of collapsed reads, since both ends of these are informative for PCR duplicate filtering. Described here.

Modifies AdapterRemoval parameters: --preserve5p

--mergedonly

Specify that only merged reads are sent downstream for analysis.

Singletons (i.e. reads missing a pair), or un-merged reads (where there wasn’t sufficient overlap) are discarded.

You may want to use this if you want ensure only the best quality reads for your analysis, but with the penalty of potentially losing still valid data (even if some reads have slightly lower quality). It is highly recommended when using --dedupper 'dedup' (see below).

Read Mapping Parameters

If using TSV input, mapping is performed at the library level, i.e. after lane merging.

--mapper

Specify which mapping tool to use. Options are BWA aln ('bwaaln'), BWA mem ('bwamem'), circularmapper ('circularmapper'), or bowtie2 (bowtie2). BWA aln is the default and highly suited for short-read ancient DNA. BWA mem can be quite useful for modern DNA, but is rarely used in projects for ancient DNA. CircularMapper enhances the mapping procedure to circular references, using the BWA algorithm but utilizing a extend-remap procedure (see Peltzer et al 2016, Genome Biology for details). Bowtie2 is similar to BWA aln, and has recently been suggested to provide slightly better results under certain conditions (Poullet and Orlando 2020), as well as providing extra functionality (such as FASTQ trimming). Default is ‘bwaaln’

More documentation can be seen for each tool under:

• BWA aln
• BWA mem
• CircularMapper
• Bowtie2

BWA (default)

These parameters configure mapping algorithm parameters.

--bwaalnn

Defines how many mismatches from the reference are allowed in a read. By default set to 0.04 (following recommendations of Schubert et al. (2012 BMC Genomics)), if you’re uncertain what to set check out this Shiny App for more information on how to set this parameter efficiently.

Modifies bwa aln parameter: -n

--bwaalnk

Modifies the number of mismatches in the seed during the seeding phase in the bwa aln mapping algorithm. Default is set to 2.

Modifies BWA aln parameter: -k

--bwaalnl

Configures the length of the seed used during seeding. Default is set to be ‘turned off’ at the recommendation of Schubert et al. (2012 BMC Genomics) for ancient DNA with 1024.

Note: Despite being recommended, turning off seeding can result in long runtimes!

Modifies BWA aln parameter: -l

CircularMapper

--circularextension

The number of bases to extend the reference genome with. By default this is set to 500 if not specified otherwise.

Modifies circulargenerator and realignsamfile parameter: -e

--circulartarget

The chromosome in your FASTA reference that you’d like to be treated as circular. By default this is set to MT but can be configured to match any other chromosome.

Modifies circulargenerator parameter: -s

--circularfilter

If you want to filter out reads that don’t map to a circular chromosome, turn this on. By default this option is turned off.

Bowtie2

--bt2_alignmode

The type of read alignment to use. Options are ‘local’ or ‘end-to-end’. Local allows only partial alignment of read, with ends of reads possibly ‘soft-clipped’ (i.e. remain unaligned/ignored), if the soft-clipped alignment provides best alignment score. End-to-end requires all nucleotides to be aligned. Default is ‘local’, following Cahill et al (2018) and Poullet and Orlando 2020.

Modifies Bowtie2 parameters: --very-fast --fast --sensitive --very-sensitive --very-fast-local --fast-local --sensitive-local --very-sensitive-local

--bt2_sensitivity

The Bowtie2 ‘preset’ to use. Options: ‘no-preset’ ‘very-fast’, ‘fast’, ‘sensitive’, or ‘very-sensitive’. These strings apply to both --bt2_alignmode options. See the Bowtie2 manual for actual settings. Default is ‘sensitive’ (following Poullet and Orlando (2020), when running damaged-data without UDG treatment)

Modifies Bowtie2 parameters: --very-fast --fast --sensitive --very-sensitive --very-fast-local --fast-local --sensitive-local --very-sensitive-local

--bt2n

The number of mismatches allowed in the seed during seed-and-extend procedure of Bowtie2. This will override any values set with --bt2_sensitivity. Can either be 0 or 1. Default: 0 (i.e. use--bt2_sensitivity defaults).

Modifies Bowtie2 parameters: -N

--bt2l

The length of the seed sub-string to use during seeding. This will override any values set with --bt2_sensitivity. Default: 0 (i.e. use--bt2_sensitivity defaults: 20 for local and 22 for end-to-end.

Modifies Bowtie2 parameters: -L

-bt2_trim5

Number of bases to trim at the 5’ (left) end of read prior alignment. Maybe useful when left-over sequencing artefacts of in-line barcodes present Default: 0

Modifies Bowtie2 parameters: -bt2_trim5

-bt2_trim3

Number of bases to trim at the 3’ (right) end of read prior alignment. Maybe useful when left-over sequencing artefacts of in-line barcodes present Default: 0.

Modifies Bowtie2 parameters: -bt2_trim3

Removal of Host-Mapped Reads

These parameters are used for removing mapped reads from the original input FASTQ files, usually in the context of uploading the original FASTQ files to a public read archive (NCBI SRA/EBI ENA/DDBJ SRA).

These flags will produce FASTQ files almost identical to your input files, except that reads with the same read ID as one found in the mapped bam file, are either removed or ‘masked’ (every base replaced with Ns).

This functionality allows you to provide other researchers who wish to re-use your data to apply their own adapter removal/read merging procedures, while maintaining anonymity for sample donors - for example with microbiome research.

If using TSV input, mapped read removal is performed per library, i.e. after lane merging.

--hostremoval_input_fastq

Create pre-Adapter Removal FASTQ files without reads that mapped to reference (e.g. for public upload of privacy sensitive non-host data)

--hostremoval_mode

Read removal mode. Completely remove mapped reads from the file(s) ('remove') or just replace mapped reads sequence by N ('replace')

Modifies extract_map_reads.py parameter: -m

Read Filtering and Conversion Parameters

Users can configure to keep/discard/extract certain groups of reads efficiently in the nf-core/eager pipeline.

If using TSV input, filtering is performed library, i.e. after lane merging.

This module utilises samtools view and filter_bam_fragment_length.py

--run_bam_filtering

Turns on the bam filtering module for either mapping quality filtering or unmapped read treatment.

⚠️ this is required for metagenomic screening!

--bam_mapping_quality_threshold

Specify a mapping quality threshold for mapped reads to be kept for downstream analysis. By default keeps all reads and is therefore set to 0 (basically doesn’t filter anything).

Modifies samtools view parameter: -q

--bam_filter_minreadlength

Specify minimum length of mapped reads. This filtering will apply at the same time as mapping quality filtering.

If used instead of minimum length read filtering at AdapterRemoval, this can be useful to get more realistic endogenous DNA percentages, when most of your reads are very short (e.g. in single-stranded libraries) and would otherwise be discarded by AdapterRemoval (thus making an artificially small denominator for a typical endogenous DNA calculation). Note in this context you should not perform mapping quality filtering nor discarding of unmapped reads to ensure a correct denominator of all reads, for the endogenous DNA calculation.

Modifies filter_bam_fragment_length.py parameter: -l

--bam_unmapped_type

Defines how to proceed with unmapped reads: 'discard' removes all unmapped reads, keep keeps both unmapped and mapped reads in the same BAM file, 'bam' keeps unmapped reads as BAM file, 'fastq' keeps unmapped reads as FastQ file, both keeps both BAM and FASTQ files. Default is discard. keep is what would happen if --run_bam_filtering was not supplied.

Note that in all cases, if --bam_mapping_quality_threshold is also supplied, mapping quality filtering will still occur on the mapped reads.

⚠️ --bam_unmapped_type 'fastq' is required for metagenomic screening!

Modifies samtools view parameter: -f4 -F4

Read DeDuplication Parameters

If using TSV input, deduplication is performed per library, i.e. after lane merging.

--dedupper

Sets the duplicate read removal tool. By default uses markduplicates from Picard. Alternatively an ancient DNA specific read deduplication tool dedup (Peltzer et al. 2016) is offered.

This utilises both ends of paired-end data to remove duplicates (i.e. true exact duplicates, as markduplicates will over-zealously deduplicate anything with the same starting position even if the ends are different). DeDup should only be used solely on paired-end data otherwise suboptimal deduplication can occur if applied to either single-end or a mix of single-end/paired-end data.

Note that if you run without the --mergedonly flag for AdapterRemoval, DeDup will likely fail. If you absolutely want to use both PE and SE data, you can supply the --dedup_all_merged flag to consider singletons to also be merged paired-end reads. This may result in over-zealous deduplication.

--dedup_all_merged

Sets DeDup to treat all reads as merged reads. This is useful if reads are for example not prefixed with M_ in all cases. Therefore, this can be used as a workaround when also using a mixture of paired-end and single-end data, however this is not recommended (see above).

Modifies dedup parameter: -m

Library Complexity Estimation Parameters

nf-core/eager uses Preseq on mapped reads as one method to calculate library complexity. If DeDup is used, Preseq uses the histogram output of DeDup, otherwise the sorted non-duplicated BAM file is supplied. Furthermore, if paired-end read collapsing is not performed, the -P flag is used.

--preseq_step_size

Can be used to configure the step size of Preseq’s c_curve method. Can be useful when only few and thus shallow sequencing results are used for extrapolation.

Modifies preseq c_curve parameter: -s

DNA Damage Assessment Parameters

More documentation can be seen in the follow links for:

• DamageProfiler
• PMDTools documentation

If using TSV input, DamageProfiler is performed per library, i.e. after lane merging. PMDtools and BAM Trimming is run after library merging of same-named library BAMs that have the same type of UDG treatment. BAM Trimming is only performed on non-UDG and half-UDG treated data.

--damageprofiler_length

Specifies the length filter for DamageProfiler. By default set to 100.

Modifies DamageProfile parameter: -l

--damageprofiler_threshold

Specifies the length of the read start and end to be considered for profile generation in DamageProfiler. By default set to 15 bases.

Modifies DamageProfile parameter: -t

--damageprofiler_yaxis

Specifies what the maximum misincorporation frequency should be displayed as, in the DamageProfiler damage plot. This is set to 0.30 (i.e. 30%) by default as this matches the popular mapDamage2.0 program. However, the default behaviour of DamageProfiler is to ‘autoscale’ the y-axis maximum to zoom in on any possible damage that may occur (e.g. if the damage is about 10%, the highest value on the y-axis would be set to 0.12). This ‘autoscale’ behaviour can be turned on by specifying the number to 0. Default: 0.30.

Modifies DamageProfile parameter: -yaxis_damageplot

--run_pmdtools

Specifies to run PMDTools for damage based read filtering and assessment of DNA damage in sequencing libraries. By default turned off.

--pmdtools_range

Specifies the range in which to consider DNA damage from the ends of reads. By default set to 10.

Modifies PMDTools parameter: --range

--pmdtools_threshold

Specifies the PMDScore threshold to use in the pipeline when filtering BAM files for DNA damage. Only reads which surpass this damage score are considered for downstream DNA analysis. By default set to 3 if not set specifically by the user.

Modifies PMDTools parameter: --threshold

--pmdtools_reference_mask

Can be used to set a path to a reference genome mask for PMDTools.

--pmdtools_max_reads

The maximum number of reads used for damage assessment in PMDtools. Can be used to significantly reduce the amount of time required for damage assessment in PMDTools. Note that a too low value can also obtain incorrect results.

Modifies PMDTools parameter: -n

Feature Annotation Statistics

If you’re interested in looking at coverage stats for certain features on your reference such as genes, SNPs etc., you can use the following bedtools module for this purpose.

More documentation on bedtools can be seen in the bedtools documentation

If using TSV input, bedtools is run after library merging of same-named library BAMs that have the same type of UDG treatment.

--run_bedtools_coverage

Specifies to turn on the bedtools module, producing statistics for breadth (or percent coverage), and depth (or X fold) coverages.

--anno_file

Specify the path to a GFF/BED containing the feature coordinates (or any acceptable input for bedtools coverage). Must be in quotes.

BAM Trimming Parameters

For some library preparation protocols, users might want to clip off damaged bases before applying genotyping methods. This can be done in nf-core/eager automatically by turning on the --run_trim_bam parameter.

More documentation can be seen in the bamUtil documentation

--run_trim_bam

Turns on the BAM trimming method. Trims off [n] bases from reads in the deduplicated BAM file. Damage assessment in PMDTools or DamageProfiler remains untouched, as data is routed through this independently. BAM trimming is typically performed to reduce errors during genotyping that can be caused by aDNA damage.

BAM trimming will only be performed on libraries indicated as --udg_type 'none' or --udg_type 'half'. Complete UDG treatment (‘full’) should have removed all damage. The amount of bases that will be trimmed off can be set separately for libraries with --udg_type 'none' and 'half' (see --bamutils_clip_half_udg_left / --bamutils_clip_half_udg_right / --bamutils_clip_none_udg_left / --bamutils_clip_none_udg_right).

Note: additional artefacts such as bar-codes or adapters that could potentially also be trimmed should be removed prior mapping.

Modifies bamUtil’s trimBam parameter: -L -R

--bamutils_clip_half_udg_left / --bamutils_clip_half_udg_right

Default set to 1 and clips off one base of the left or right side of reads from libraries whose UDG treatment is set to half. Note that reverse reads will automatically be clipped off at the reverse side with this (automatically reverses left and right for the reverse read).

Modifies bamUtil’s trimBam parameter: -L -R

--bamutils_softclip

By default, nf-core/eager uses hard clipping and sets clipped bases to N with quality ! in the BAM output. Turn this on to use soft-clipping instead, masking reads at the read ends respectively using the CIGAR string.

Modifies bamUtil’s trimBam parameter: -c

Genotyping Parameters

There are options for different genotypers (or genotype likelihood calculators) to be used. We suggest you read the documentation of each tool to find the ones that suit your needs.

Documentation for each tool:

• GATK UnifiedGenotyper
• GATK HaplotypeCaller
• FreeBayes
• ANGSD
• sequenceTools pileupCaller

If using TSV input, genotyping is performed per sample (i.e. after all types of libraries are merged), except for pileupCaller which gathers all double-stranded and single-stranded (same-type merged) libraries respectively.

--run_genotyping

Turns on genotyping to run on all post-dedup and downstream BAMs. For example if --run_pmdtools and --trim_bam are both supplied, the genotyper will be run on all three BAM files i.e. post-deduplication, post-pmd and post-trimmed BAM files.

--genotyping_tool

Specifies which genotyper to use. Current options are: GATK (v3.5) UnifiedGenotyper or GATK Haplotype Caller (v4); and the FreeBayes Caller. Specify ‘ug’, ‘hc’, ‘freebayes’, ‘pileupcaller’ and ‘angsd’ respectively.

Note that while UnifiedGenotyper is more suitable for low-coverage ancient DNA (HaplotypeCaller does de novo assembly around each variant site), it is officially deprecated by the Broad Institute and is only accessible by an archived version not properly available on conda. Therefore if specifying ‘ug’, will need to supply a GATK 3.5 -jar to the parameter gatk_ug_jar. Note that this means the pipeline is not fully reproducible in this configuration, unless you personally supply the .jar file.

--genotyping_source

Indicates which BAM file to use for genotyping, depending on what BAM processing modules you have turned on. Options are: 'raw' for mapped only, filtered, or DeDup BAMs (with priority right to left); 'trimmed' (for base clipped BAMs); 'pmd' (for pmdtools output). Default is: 'raw'.

--gatk_ug_jar

Specify a path to a local copy of a GATK 3.5 .jar file, preferably version ‘3.5-0-g36282e4’. The download location of this may be available from the GATK forums or the Google Cloud Storage of the Broad Institute.

You must manually report your version of GATK 3.5 in publications/MultiQC as it is not included in our container.

--gatk_call_conf

If selected, specify a GATK genotyper phred-scaled confidence threshold of a given SNP/INDEL call. Default: 30

Modifies GATK UnifiedGenotyper or HaplotypeCaller parameter: -stand_call_conf

--gatk_ploidy

If selected, specify a GATK genotyper ploidy value of your reference organism. E.g. if you want to allow heterozygous calls from >= diploid organisms. Default: 2

Modifies GATK UnifiedGenotyper or HaplotypeCaller parameter: --sample-ploidy

--gatk_downsample

Maximum depth coverage allowed for genotyping before down-sampling is turned on. Any position with a coverage higher than this value will be randomly down-sampled to 250 reads. Default: 250

Modifies GATK UnifiedGenotyper parameter: -dcov

--gatk_dbsnp

(Optional) Specify VCF file for output VCF SNP annotation e.g. if you want to annotate your VCF file with ‘rs’ SNP IDs. Check GATK documentation for more information. Gzip not accepted.

--gatk_hc_out_mode

If the GATK genotyper HaplotypeCaller is selected, what type of VCF to create, i.e. produce calls for every site or just confidence sites. Options: 'EMIT_VARIANTS_ONLY', 'EMIT_ALL_CONFIDENT_SITES', 'EMIT_ALL_ACTIVE_SITES'. Default: 'EMIT_VARIANTS_ONLY'

Modifies GATK HaplotypeCaller parameter: -output_mode

--gatk_hc_emitrefconf

If the GATK HaplotypeCaller is selected, mode for emitting reference confidence calls. Options: 'NONE', 'BP_RESOLUTION', 'GVCF'. Default: 'GVCF'

Modifies GATK HaplotypeCaller parameter: --emit-ref-confidence

--gatk_ug_out_mode

If the GATK UnifiedGenotyper is selected, what type of VCF to create, i.e. produce calls for every site or just confidence sites. Options: 'EMIT_VARIANTS_ONLY', 'EMIT_ALL_CONFIDENT_SITES', 'EMIT_ALL_SITES'. Default: 'EMIT_VARIANTS_ONLY'

Modifies GATK UnifiedGenotyper parameter: --output_mode

--gatk_ug_genotype_model

If the GATK UnifiedGenotyper is selected, which likelihood model to follow, i.e. whether to call use SNPs or INDELS etc. Options: 'SNP', 'INDEL', 'BOTH', 'GENERALPLOIDYSNP', 'GENERALPLOIDYINDEL’. Default: 'SNP'

Modifies GATK UnifiedGenotyper parameter: --genotype_likelihoods_model

--gatk_ug_keep_realign_bam

If provided when running GATK’s UnifiedGenotyper, this will put the BAMs into the output folder, that have realigned reads (with GATK’s (v3) IndelRealigner) around possible variants for improved genotyping.

These BAMs will be stored in the same folder as the corresponding VCF files.

--gatk_ug_gatk_ug_defaultbasequalities

When running GATK’s UnifiedGenotyper, specify a value to set base quality scores, if reads are missing this information. Might be useful if you have ‘synthetically’ generated reads (e.g. chopping up a reference genome). Default is set to -1 which is to not set any default quality (turned off). Default: -1

Modifies GATK UnifiedGenotyper parameter: --defaultBaseQualities

--freebayes_C

Specify minimum required supporting observations to consider a variant. Default: 1

Modifies freebayes parameter: -C

--freebayes_g

Specify to skip over regions of high depth by discarding alignments overlapping positions where total read depth is greater than specified C. Not set by default.

Modifies freebayes parameter: -g

--freebayes_p

Specify ploidy of sample in FreeBayes. Default is diploid. Default: 2

Modifies freebayes parameter: -p

--pileupcaller_bedfile

Specify a SNP panel in the form of a bed file of sites at which to generate pileup for pileupCaller.

--pileupcaller_snpfile

Specify a SNP panel in EIGENSTRAT format, pileupCaller will call these sites.

--pileupcaller_method

Specify calling method to use. Options: randomHaploid, randomDiploid, majorityCall. Default: 'randomHaploid'

Modifies pileupCaller parameter: --randomHaploid --randomDiploid --majorityCall

--pileupcaller_transitions_mode

Specify if genotypes of transition SNPs should be called, set to missing, or excluded from the genotypes respectively. Options: 'AllSites', 'TransitionsMissing', 'SkipTransitions'. Default: 'AllSites'

Modifies pileupCaller parameter: --skipTransitions --transitionsMissing

--angsd_glmodel

Specify which genotype likelihood model to use. Options: 'samtools, 'gatk', 'soapsnp', 'syk'. Default: 'samtools'

Modifies ANGSD parameter: -GL

--angsd_glformat

Specifies what type of genotyping likelihood file format will be output. Options: 'text', 'binary', 'binary_three', 'beagle_binary'. Default: 'text'.

The options refer to the following descriptions respectively:

• text: textoutput of all 10 log genotype likelihoods.
• binary: binary all 10 log genotype likelihood
• binary_three: binary 3 times likelihood
• beagle_binary: beagle likelihood file

See the ANGSD documentation for more information on which to select for your downstream applications.

Modifies ANGSD parameter: -doGlF

--angsd_createfasta

Turns on the ANGSD creation of a FASTA file from the BAM file.

--angsd_fastamethod

The type of base calling to be performed when creating the ANGSD FASTA file. Options: 'random' or 'common'. Will output the most common non-N base at each given position, whereas ‘random’ will pick one at random. Default: 'random'.

Modifies ANGSD parameter: -doFasta -doCounts

Consensus Sequence Generation

If using TSV input, consensus generation is performed per sample (i.e. after all types of libraries are merged).

--run_vcf2genome

Turn on consensus sequence genome creation via VCF2Genome. Only accepts GATK UnifiedGenotyper VCF files with the --gatk_ug_out_mode 'EMIT_ALL_SITES' and --gatk_ug_genotype_model 'SNP flags. Typically useful for small genomes such as mitochondria.

--vcf2genome_outfile

The name of your requested output FASTA file. Do not include .fasta suffix.

--vcf2genome_header

The name of the FASTA entry you would like in your FASTA file.

--vcf2genome_minc

Minimum depth coverage for a SNP to be made. Else, a SNP will be called as N. Default: 5

Modifies VCF2Genome parameter: -minc

--vcf2genome_minq

Minimum genotyping quality of a call to be made. Else N will be called. Default: 30

Modifies VCF2Genome parameter: -minq

--vcf2genome_minfreq

In the case of two possible alleles, the frequency of the majority allele required for a call to be made. Else, a SNP will be called as N. Default: 0.8

Modifies VCF2Genome parameter: -minfreq

SNP Table Generation

SNP Table Generation here is performed by MultiVCFAnalyzer. The current version of MultiVCFAnalyzer version only accepts GATK UnifiedGenotyper 3.5 VCF files, and when the ploidy was set to 2 (this allows MultiVCFAnalyzer to report frequencies of polymorphic positions). A description of how the tool works can be seen in the Supplementary Information of Bos et al. (2014) under “SNP Calling and Phylogenetic Analysis”.

More can be seen in the MultiVCFAnalyzer documentation.

If using TSV input, MultiVCFAnalyzer is performed on all samples gathered together.

--run_multivcfanalyzer

Turns on MultiVCFAnalyzer. Will only work when in combination with UnifiedGenotyper genotyping module (see --genotyping_tool).

--write_allele_frequencies

Specify whether to tell MultiVCFAnalyzer to write within the SNP table the frequencies of the allele at that position e.g. A (70%).

--min_genotype_quality

The minimal genotyping quality for a SNP to be considered for processing by MultiVCFAnalyzer. The default threshold is 30.

--min_base_coverage

The minimal number of reads covering a base for a SNP at that position to be considered for processing by MultiVCFAnalyzer. The default depth is 5.

--min_allele_freq_hom

The minimal frequency of a nucleotide for a ‘homozygous’ SNP to be called. In other words, e.g. 90% of the reads covering that position must have that SNP to be called. If the threshold is not reached, and the previous two parameters are matched, a reference call is made (displayed as . in the SNP table). If the above two parameters are not met, an ‘N’ is called. The default allele frequency is 0.9.

--min_allele_freq_het

The minimum frequency of a nucleotide for a ‘heterozygous’ SNP to be called. If this parameter is set to the same as --min_allele_freq_hom, then only homozygous calls are made. If this value is less than the previous parameter, then a SNP call will be made. If it is between this and the previous parameter, it will be displayed as a IUPAC uncertainty call. Default is 0.9.

--additional_vcf_files

If you wish to add to the table previously created VCF files, specify here a path with wildcards (in quotes). These VCF files must be created the same way as your settings for GATK UnifiedGenotyping module above.

--reference_gff_annotations

If you wish to report in the SNP table annotation information for the regions SNPs fall in, provide a file in GFF format (the path must be in quotes).

--reference_gff_exclude

If you wish to exclude SNP regions from consideration by MultiVCFAnalyzer (such as for problematic regions), provide a file in GFF format (the path must be in quotes).

--snp_eff_results

If you wish to include results from SNPEff effect analysis, supply the output from SNPEff in txt format (the path must be in quotes).

Mitochondrial to Nuclear Ratio

If using TSV input, Mitochondrial to Nuclear Ratio calculation is calculated per deduplicated library (after lane merging)

--run_mtnucratio

Turn on the module to estimate the ratio of mitochondrial to nuclear reads.

--mtnucratio_header

Specify the FASTA entry in the reference file specified as --fasta, which acts as the mitochondrial ‘chromosome’ to base the ratio calculation on. The tool only accepts the first section of the header before the first space. The default chromosome name is based on hs37d5/GrCH37 human reference genome. Default: ‘MT’

Human Sex Determination

An optional process for human DNA. It can be used to calculate the relative coverage of X and Y chromosomes compared to the autosomes (X-/Y-rate). Standard errors for these measurements are also calculated, assuming a binomial distribution of reads across the SNPs.

If using TSV input, SexDetERRmine is performed on all samples gathered together.

--run_sexdeterrmine

Specify to run the optional process of sex determination.

--sexdeterrmine_bedfile

Specify an optional bedfile of the list of SNPs to be used for X-/Y-rate calculation. Running without this parameter will considerably increase runtime, and render the resulting error bars untrustworthy. Theoretically, any set of SNPs that are distant enough that two SNPs are unlikely to be covered by the same read can be used here. The programme was coded with the 1240K panel in mind. The path must be in quotes.

Human Nuclear Contamination

--run_nuclear_contamination

Specify to run the optional processes for human nuclear DNA contamination estimation.

--contamination_chrom_name

The name of the chromosome X in your bam. 'X' for hs37d5, 'chrX' for HG19. Defaults to 'X'.

Metagenomic Screening

An increasingly common line of analysis in high-throughput aDNA analysis today is simultaneously screening off target reads of the host for endogenous microbial signals - particularly of pathogens. Metagenomic screening is currently offered via MALT with aDNA specific verification via MaltExtract, or Kraken2.

Please note the following:

• ⚠️ Metagenomic screening is only performed on unmapped reads from a mapping step.
  • You must supply the --run_bam_filtering flag with unmapped reads in FASTQ format.
  • If you wish to run solely MALT (i.e. the HOPS pipeline), you must still supply a small decoy genome like phiX or human mtDNA --fasta.
• MALT database construction functionality is not included within the pipeline
  • this should be done independently, prior the nf-core/eager run.
  • To use malt-build from the same version as malt-run, load either the Docker, Singularity or Conda environment.
• MALT can often require very large computing resources depending on your database. We set a absolute minimum of 16 cores and 128GB of memory (which is 1/4 of the recommendation from the developer). Please leave an issue on the nf-core github if you would like to see this changed.

⚠️ Running MALT on a server with less than 128GB of memory should be performed at your own risk.

If using TSV input, metagenomic screening is performed on all samples gathered together.

--run_metagenomic_screening

Turn on the metagenomic screening module.

--metagenomic_tool

Specify which taxonomic classifier to use. There are two options available:

• kraken for Kraken2
• malt for MALT

⚠️ Important It is very important to run nextflow clean -f on your Nextflow run directory once completed. RMA6 files are VERY large and are copied from a work/ directory into the results folder. You should clean the work directory with the command to ensure non-redundancy and large HDD footprints!

--database

Specify the path to the directory containing your taxonomic classifier’s database (malt or kraken).

For Kraken2, it can be either the path to the directory or the path to the .tar.gz compressed directory of the Kraken2 database.

--metagenomic_min_support_reads

Specify the minimum number of reads a given taxon is required to have to be retained as a positive ‘hit’.
For malt, this only applies when --malt_min_support_mode is set to ‘reads’. Default: 1.

Modifies MALT or kraken_parse.py parameter: -sup and -c respectively

--percent_identity

Specify the minimum percent identity (or similarity) a sequence must have to the reference for it to be retained. Default is 85

Only used when --metagenomic_tool malt is also supplied.

Modifies MALT parameter: -id

--malt_mode

Use this to run the program in ‘BlastN’, ‘BlastP’, ‘BlastX’ modes to align DNA and DNA, protein and protein, or DNA reads against protein references respectively. Ensure your database matches the mode. Check the MALT manual for more details. Default: 'BlastN'

Only when --metagenomic_tool malt is also supplied.

Modifies MALT parameter: -m

--malt_alignment_mode

Specify what alignment algorithm to use. Options are ‘Local’ or ‘SemiGlobal’. Local is a BLAST like alignment, but is much slower. Semi-global alignment aligns reads end-to-end. Default: 'SemiGlobal'

Only when --metagenomic_tool malt is also supplied.

Modifies MALT parameter: -at

--malt_top_percent

Specify the top percent value of the LCA algorithm. From the MALT manual: “For each read, only those matches are used for taxonomic placement whose bit disjointScore is within 10% of the best disjointScore for that read.”. Default: 1.

Only when --metagenomic_tool malt is also supplied.

Modifies MALT parameter: -top

--malt_min_support_mode

Specify whether to use a percentage, or raw number of reads as the value used to decide the minimum support a taxon requires to be retained.

Only when --metagenomic_tool malt is also supplied.

Modifies MALT parameter: -sup -supp

--malt_min_support_percent

Specify the minimum number of reads (as a percentage of all assigned reads) a given taxon is required to have to be retained as a positive ‘hit’ in the RMA6 file. This only applies when --malt_min_support_mode is set to ‘percent’. Default 0.01.

Only when --metagenomic_tool malt is also supplied.

Modifies MALT parameter: -supp

--malt_max_queries

Specify the maximum number of alignments a read can have. All further alignments are discarded. Default: 100

Only when --metagenomic_tool malt is also supplied.

Modifies MALT parameter: -mq

--malt_memory_mode

How to load the database into memory. Options are 'load', 'page' or 'map'. ‘load’ directly loads the entire database into memory prior seed look up, this is slow but compatible with all servers/file systems. 'page' and 'map' perform a sort of ‘chunked’ database loading, allowing seed look up prior entire database loading. Note that Page and Map modes do not work properly not with many remote file-systems such as GPFS. Default is 'load'.

Only when --metagenomic_tool malt is also supplied.

Modifies MALT parameter: --memoryMode

--malt_sam_output

Specify to also produce gzipped SAM files of all alignments and un-aligned reads in addition to RMA6 files. These are not soft-clipped or in ‘sparse’ format. Can be useful for downstream analyses due to more common file format.

⚠️ can result in very large run output directories as this is essentially duplication of the RMA6 files.

Modifies MALT parameter -a -f

Metagenomic Authentication

--run_maltextract

Turn on MaltExtract for MALT aDNA characteristics authentication of metagenomic output from MALT.

More can be seen in the MaltExtract documentation

Only when --metagenomic_tool malt is also supplied

--maltextract_taxon_list

Path to a .txt file with taxa of interest you wish to assess for aDNA characteristics. In .txt file should be one taxon per row, and the taxon should be in a valid NCBI taxonomy name format.

Only when --metagenomic_tool malt is also supplied.

--maltextract_ncbifiles

Path to directory containing containing the NCBI resource tree and taxonomy table files (ncbi.tre and ncbi.map; available at the HOPS repository).

Only when --metagenomic_tool malt is also supplied.

--maltextract_filter

Specify which MaltExtract filter to use. This is used to specify what types of characteristics to scan for. The default will output statistics on all alignments, and then a second set with just reads with one C to T mismatch in the first 5 bases. Further details on other parameters can be seen in the HOPS documentation. Options: 'def_anc', 'ancient', 'default', 'crawl', 'scan', 'srna', ‘assignment’. Default: 'def_anc'.

Only when --metagenomic_tool malt is also supplied.

Modifies MaltExtract parameter: -f

--maltextract_toppercent

Specify frequency of top alignments for each read to be considered for each node. Default is 0.01, i.e. 1% of all reads (where 1 would correspond to 100%).

⚠️ this parameter follows the same concept as --malt_top_percent but uses a different notation i.e. integer (MALT) versus float (MALTExtract)

Default: 0.01.

Only when --metagenomic_tool malt is also supplied.

Modifies MaltExtract parameter: -a

--maltextract_destackingoff

Turn off destacking. If left on, a read that overlaps with another read will be removed (leaving a depth coverage of 1).

Only when --metagenomic_tool malt is also supplied.

Modifies MaltExtract parameter: --destackingOff

--maltextract_downsamplingoff

Turn off downsampling. By default, downsampling is on and will randomly select 10,000 reads if the number of reads on a node exceeds this number. This is to speed up processing, under the assumption at 10,000 reads the species is a ‘true positive’.

Only when --metagenomic_tool malt is also supplied.

Modifies MaltExtract parameter: --downSampOff

--maltextract_duplicateremovaloff

Turn off duplicate removal. By default, reads that are an exact copy (i.e. same start, stop coordinate and exact sequence match) will be removed as it is considered a PCR duplicate.

Only when --metagenomic_tool malt is also supplied.

Modifies MaltExtract parameter: --dupRemOff

--maltextract_matches

Export alignments of hits for each node in BLAST format. By default turned off.

Only when --metagenomic_tool malt is also supplied.

Modifies MaltExtract parameter: --matches

--maltextract_megansummary

Export ‘minimal’ summary files (i.e. without alignments) that can be loaded into MEGAN6. By default turned off.

Only when --metagenomic_tool malt is also supplied.

Modifies MaltExtract parameter: --meganSummary

--maltextract_percentidentity

Minimum percent identity alignments are required to have to be reported. Higher values allows fewer mismatches between read and reference sequence, but therefore will provide greater confidence in the hit. Lower values allow more mismatches, which can account for damage and divergence of a related strain/species to the reference. Recommended to set same as MALT parameter or higher. Default: 85.0.

Only when --metagenomic_tool malt is also supplied.

Modifies MaltExtract parameter: --minPI

maltextract_topalignment

Use the best alignment of each read for every statistic, except for those concerning read distribution and coverage. Default: off.

Only when --metagenomic_tool malt is also supplied.

Modifies MaltExtract parameter: --useTopAlignment

Clean up

Once a run has completed, you will have lots of (some very large) intermediate files in your output directory. These are stored within the directory named work.

After you have verified your run completed correctly and everything in the module output directories are present as you expect and need, you can perform a clean-up.

Important: Once clean-up is completed, you will not be able to re-rerun the pipeline from an earlier step and you’ll have to re-run from scratch.

While in your output directory, firstly verify you’re only deleting files stored in work/ with the dry run command:

nextflow clean -n

⚠️ some institutional profiles already have clean-up on successful run completion turned on by default.

If you’re ready, you can then remove the files with

nextflow clean -f -k

This will make your system administrator very happy as you will halve the hard drive footprint of the run, so be sure to do this!

Troubleshooting and FAQs

My pipeline update doesn’t seem to do anything

To download a new version of a pipeline, you can use the following, replacing <VERSION> to the corresponding version.

nextflow pull nf-core/eager -r <VERSION>

However, in very rare cases, minor fixes to a version will be pushed out without a version number bump. This can confuse nextflow slightly, as it thinks you already have the ‘broken’ version from your original pipeline download.

If when running the pipeline you don’t see any changes in the fixed version when running it, you can try removing your nextflow EAGER cache typically stored in your home directory with

rm -r ~/.nextflow/assets/nf-core/eager

And re-pull the pipeline with the command above. This will install a fresh version of the version with the fixes.

Input files not found

When using the direct input method: if no file, only one input file, or only ‘read one’ and not ‘read two’ is picked up then something is likely wrong with your input file declaration (--input):

1. The path must be enclosed in quotes (' or ")
2. The path must have at least one * wildcard character. This is even if you are only running one paired end sample.
3. When using the pipeline with paired end data, the path must use {1,2} or {R1,R2} notation to specify read pairs.
4. If you are running single-end data make sure to specify --single_end

Important: The pipeline can’t take a list of multiple input files when using the direct input method - it takes a ‘glob’ expression. If your input files are scattered in different paths then we recommend that you generate a directory with symlinked files. If running in paired-end mode please make sure that your files are sensibly named so that they can be properly paired. See the previous point.

If the pipeline can’t find your files then you will get the following error

ERROR ~ Cannot find any reads matching: *{1,2}.fastq.gz

If your sample name is “messy” then you have to be very particular with your glob specification. A file name like L1-1-D-2h_S1_L002_R1_001.fastq.gz can be difficult enough for a human to read. Specifying *{1,2}*.gz won’t work give you what you want whilst *{R1,R2}*.gz (i.e. the addition of the Rs) will.

If using the TSV input method, this likely means there is a mistake or typo in the path in a given column. Often this is a trailing space at the end of the path.

I am only getting output for a single sample although I specified multiple with wildcards

You must specify paths to files in quotes, otherwise your shell will evaluate any wildcards (*) rather than Nextflow.

For example

nextflow run nf-core/eager --input /path/to/sample_*/*.fq.gz

Would be evaluated by your shell as

nextflow run nf-core/eager --input /path/to/sample_1/sample_1.fq.gz /path/to/sample_1/sample_1.fq.gz /path/to/sample_1/sample_1.fq.gz

And Nextflow will only take the first path after --input, ignoring the others.

On the other hand, encapsulating the path in quotes will allow Nextflow to evaluate the paths.

nextflow run nf-core/eager --input "/path/to/sample_*/*.fq.gz"

The pipeline crashes almost immediately with an early pipeline step

Sometimes a newly downloaded and set up nf-core/eager pipeline will encounter an issue where a run almost immediately crashes (e.g. at fastqc, output_documentation etc.) saying the tool could not be found or similar.

I am running Docker

You may have an outdated container. This happens more often when running on the dev branch of nf-core/eager, because Docker will not update the container on each new commit, and thus may not get new tools called within the pipeline code.

To fix, just re-pull the nf-core/eager Docker container manually with:

docker pull nfcore/eager:dev

I am running Singularity

If you’re running Singularity, it could be that Nextflow cannot access your Singularity image properly - often due to missing bind paths.

See here for more information.

The pipeline has crashed with an error but Nextflow is still running

If this happens, you can either wait until all other already running jobs to safely finish, or if Nextflow still does not stop press ctrl + c on your keyboard (or equivalent) to stop the Nextflow run.

⚠️ if you do this, and do not plan to fix the run make sure to delete the output folder. Otherwise you may end up a lot of large intermediate files being left! You can clean a Nextflow run of all intermediate files with nextflow clean -f -k or delete the work/ directory.

I get a exceeded job memory limit error

While Nextflow tries to make your life easier by automatically retrying jobs that run out of memory with more resources (until your specified max-limit), sometimes you may have such large data you run out even after the default 3 retries.

To fix this you need to change the default memory requirements for the process that is breaking. We can do this by making a custom profile, which we then provide to the Nextflow run command.

For example, lets say it’s the markduplicates process that is running out of memory.

First we need to check to see what default memory value we have. We can do this by going to the main nf-core/eager code and opening the main.nf file. We can then use your browser’s find functionality for: process markduplicates.

Once found, we then need to check the line called label. In this case the label is mc_small (for multi-core small).

Next we need to go back to the main github repository, and open conf/base.config. Again using our find functionality, we search for: withLabel:'mc_small'.

We see that the memory is set to 4.GB (memory = { check_max( 4.GB * task.attempt, 'memory' )})).

Now back on your computer, we need to make a new file called custom_resources.conf. You should save it somewhere centrally so you can reuse it.

If you think this would be useful for multiple people in your lab/institute, we highly recommend you make an institutional profile at nf-core/configs. This will simplify this process in the future.

Within this file, you will need to add the following:

profiles {
    big_data {
      process {
        withName: markduplicates {
          memory = { check_max( 16.GB * task.attempt, 'memory' ) }
        }
      }
    }
}

Where we have increased the default 4.GB to 16.GB. Make sure that you keep the check_max function, as this prevents your run asking for too much memory during retries.

Once saved, we can then modify your original Nextflow run command:

nextflow run nf-core/eager -r 2.2.0 -c /<path>/<to>/custom_resources.conf -profile big_data,<original>,<profiles> <...>

Where we have added -c to specify which file to use for the custom profiles, and then added the big_data profile to the original profiles you were using.

⚠️ it’s important that big_data comes first, to ensure it overwrites any parameters set in the subsequent profiles!

I get a file name collision error during merging

When using TSV input, nf-core/eager will attempt to merge all Lanes of a Library_ID, or all files with the same Library_ID or Sample_ID. However, if you have specified the same Lane or Library_ID for two sets of FASTQ files you will likely receive an error such as

Error executing process > 'library_merge (JK2782)'
Caused by:
  Process `library_merge` input file name collision -- There are multiple input files for each of the following file names: JK2782.mapped_rmdup.bam.csi, JK2782.mapped_rmdup.bam
Tip: you can try to figure out what's wrong by changing to the process work dir and showing the script file named `.command.sh`
Execution cancelled -- Finishing pending tasks before exit

In this case: for lane merging errors, you can give ‘fake’ lane IDs to ensure they are unique (e.g. if one library was sequenced on Lane 8 of two HiSeq runs, specify lanes as 8 and 16 for each FASTQ file respectively). For library merging errors, you must modify your Library_IDs accordingly, to make them unique.

I specified a module and it didn’t produce the expected output

Possible options:

1. Check there if you have a typo in the parameter name. Nextflow does not check for this
2. Check that an upstream module was turned on (if a module requires the output of a previous module, it will not be activated unless it receives the output)

I get a unable to acquire lock

Errors like the following

Unable to acquire lock on session with ID 84333844-66e3-4846-a664-b446d070f775

normally suggest a previous Nextflow run (on the same folder) was not cleanly killed by a user (e.g. using ctrl + z to hard kill a crashed run).

To fix this, you must clean the entirety of the output directory (including output files) e.g. with rm -r <output_dir>/* <output_dir>/.* and re-running from scratch.

ctrl +z is not a recommended way of killing a Nextflow job. Runs that take a long time to fail are often still running because other job submissions are still running. Nextflow will normally wait for those processes to complete before cleaning shutting down the run (to allow rerunning of a run with -resume). ctrl + c is much safer as it will tell Nextflow to stop earlier but cleanly.

Tutorials

Tutorial - How to investigate a failed run

As with most pipelines, nf-core/eager can sometimes fail either through a problem with the pipeline itself, but also sometimes through an issue of the program being run at the given step.

To help try and identify what has caused the error, you can perform the following steps before reporting the issue:

1a Nextflow reports an ‘error executing process’ with command error

Firstly, take a moment to read the terminal output that is printed by an nf-core/eager command.

When reading the following, you can see that the actual command failed. When you get this error, this would suggest that an actual program used by the pipeline has failed. This is identifiable when you get an exit status and a Command error:, the latter of which is what is reported by the failed program itself.

ERROR ~ Error executing process > 'circulargenerator (hg19_complete_500.fasta)'
 
Caused by:
  Process `circulargenerator (hg19_complete_500.fasta)` terminated with an error exit status (1)
 
Command executed:
 
  circulargenerator -e 500 -i hg19_complete.fasta -s MT
  bwa index hg19_complete_500.fasta
 
Command exit status:
  1
 
Command output:
  (empty)
 
Command error:
  Exception in thread "main" java.lang.OutOfMemoryError: Java heap space
        at java.util.Arrays.copyOf(Arrays.java:3332)
        at java.lang.AbstractStringBuilder.ensureCapacityInternal(AbstractStringBuilder.java:124)
        at java.lang.AbstractStringBuilder.append(AbstractStringBuilder.java:448)
        at java.lang.StringBuffer.append(StringBuffer.java:270)
        at CircularGenerator.extendFastA(CircularGenerator.java:155)
        at CircularGenerator.main(CircularGenerator.java:119)
 
Work dir:
  /projects1/microbiome_calculus/RIII/03-preprocessing/mtCap_preprocessing/work/7f/52f33fdd50ed2593d3d62e7c74e408
 
Tip: you can replicate the issue by changing to the process work dir and entering the command `bash .command.run`
 
 -- Check '.nextflow.log' file for details

If you find it is a common error try and fix it yourself by changing your options in your nf-core/eager run - it could be a configuration error on your part. However in some cases it could be an error in the way we’ve set up the process in nf-core/eager.

To further investigate, go to step 2.

1b Nextflow reports an ‘error executing process’ with no command error

Alternatively, you may get an error with Nextflow itself. The most common one would be a ‘process fails’ and it looks like the following.

Error executing process > 'library_merge (JK2782)'
Caused by:
  Process `library_merge` input file name collision -- There are multiple input files for each of the following file names: JK2782.mapped_rmdup.bam.csi, JK2782.mapped_rmdup.bam
Tip: you can try to figure out what's wrong by changing to the process work dir and showing the script file named `.command.sh`
Execution cancelled -- Finishing pending tasks before exit

However in this case, there is no exit status or Command error: message. In this case this is a Nextflow issue.

The example above is because a user has specified multiple sequencing runs of different libraries but with the same library name. In this case Nextflow could not identify which is the correct file to merge because they have the same name.

This again can also be a user or Nextflow error, but the errors are often more abstract and less clear how to solve (unless you are familiar with Nextflow).

Try to investigate a bit further and see if you can understand what the error refers to, but if you cannot - please ask on the #eager channel on the nf-core slack or leave a github issue.

2 Investigating an failed process’s work/ directory

If you haven’t found a clear solution to the failed process from the reported errors, you can next go into the directory where the process was working in, and investigate the log and error messages that are produced by each command of the process.

For example, in the error in 1a you can see the following line

Work dir:
  /projects1/microbiome_calculus/RIII/03-preprocessing/mtCap_preprocessing/work/7f/52f33fdd50ed2593d3d62e7c74e408

A shortened version of the ‘hash’ directory ID can also be seen in your terminal while the pipeline is running in the square brackets at the beginning of each line.

If you change into this with cd and run ls -la you should see a collection of normal files, symbolic links (symlinks) and hidden files (indicated with . at the beginning of the file name).

• Symbolic links: are typically input files from previous processes.
• Normal files: are typically successfully completed output files from some of some of the commands in the process
• Hidden files are Nextflow generated files and include the submission commands as well as log files

When you have an error run, you can firstly check the contents of the output files to see if they are empty or not (e.g. with cat or zcat), interpretation of which will depend on the program thus dependent on the user knowledge.

Next, you can investigate .command.err and .command.out, or .command.log. These represent the standard out or error (in the case of .log, both combined) of all the commands/programs in the process - i.e. what would be printed to screen if you were running the command/program yourself. Again, view these with e.g. cat and see if you can identify the error of the program itself.

Finally, you can also try running the commands yourself. You can firstly try to do this by loading your given nf-core/eager environment (e.g. singularity shell /\<path\>/\<to\>/nf-core-eager-X-X-X.img or conda activate nf-core-eager-X.X.X), then running bash .command.sh.

If this doesn’t work, this suggests either there is something wrong with the nf-core/eager environment configuration, or there is still a problem with the program itself. To confirm the former, try running the command within the .command.sh file (viewable with cat) but with locally installed versions of programs you may already have on your system. If the command still doesn’t work, it is a problem with the program or your specified configuration. If it does work locally, please report as a github issue.

If it does, please ask the developer of the tool (although we will endeavour to help as much as we can via the nf-core slack in the #eager channel).

Tutorial - What are Profiles and How To Use Them

Tutorial Profiles - Background

A useful feature of Nextflow is the ability to use configuration profiles that can specify many default parameters and other settings on how to run your pipeline.

For example, you can use it to set your preferred mapping parameters, or specify where to keep Docker, Singularity or Conda environments, and which cluster scheduling system (and queues) your pipeline runs should normally use.

This are defined in .config files, and these in-turn can contain different profiles that can define parameters for different contexts.

For example, a .config file could contain two profiles, one for shallow-sequenced samples that uses only a small number of CPUs and memory e.g. small, and another for deep sequencing data, deep, that allows larger numbers of CPUs and memory. As another example you could define one profile called loose that contains mapping parameters to allow reads with aDNA damage to map, and then another called strict that reduces the likelihood of damaged DNA to map and cause false positive SNP calls.

Within nf-core, there are two main levels of configs

• Institutional-level profiles: these normally define things like paths to common storage, resource maximums, scheduling system
• Pipeline-level profiles: these normally define parameters specifically for a pipeline (such as mapping parameters, turning specific modules on or off)

As well as allowing more efficiency and control at cluster or Institutional levels in terms of memory usage, pipeline-level profiles can also assist in facilitating reproducible science by giving a way for researchers to ‘publish’ their exact pipeline parameters in way other users can automatically re-run the pipeline with the pipeline parameters used in the original publication but on their own cluster.

To illustrate this, lets say we analysed our data on a HPC called ‘blue’ for which an institutional profile already exists, and for our analysis we defined a profile called ‘old_dna’. We will have run our pipeline with the following command

nextflow run nf-core/eager -c old_dna_profile.config -profile old_dna,hpc_blue <...>

Then our colleague wished to recreate your results. As long as the old_dna_profile.config was published alongside your results, they can run the same pipeline settings but on their own cluster HPC ‘purple’.

nextflow run nf-core/eager -c old_dna_profile.config -profile old_dna,hpc_purple <...>

(where the old_dna profile is defined in old_dna_profile.config, and hpc_purple is defined on nf-core/configs)

This tutorial will describe how to create and use profiles that can be used by or from other researchers.

Tutorial Profiles - Inheritance Rules

Tutorial Profiles - Profiles

An important thing to understand before you start writing your own profile is understanding ‘inheritance’ of profiles when specifying multiple profiles, when using nextflow run.

When specifying multiple profiles, parameters defined in the profile in the first position will overwrite those in the second, and everything defined in the first and second will overwrite everything in a third.

This can be illustrated as follows.

           overwrites  overwrites
            ┌──────┐   ┌──────┐
            │      ▼   │      ▼
-profile my_paper,cluster,institution

This would be translated as follows.

If your parameters looked like the following

(where ‘<none>’ is a parameter not defined in a given profile.)

You can see that my_paper inherited the 0.1 parameter over the 0.01 defined in the cluster profile.

⚠️ You must always check if parameters are defined in any ‘upstream’ profiles that have been set by profile administrators that you may be unaware of. This is make sure there are no unwanted or unreported ‘defaults’ away from original nf-core/eager defaults.

Tutorial Profiles - Configuration Files

⚠️ This section is only needed for users that want to set up institutional-level profiles.

<...>
mapper = 'bwamem'
dedupper = 'markduplicates'
<...>

<...>
mapper = 'bwaaln'
<...>

nextflow run nf-core/eager -c old_dna_profile.config -profile old_dna,hpc_blue <...>

<...>
mapper = 'bwaaln'
dedupper = 'markduplicates'
<...>

// global 'fallback' parameters
params {
  // Specific nf-core/configs params
  config_profile_contact = 'James Fellows Yates (@jfy133)'
  config_profile_description = 'nf-core/eager SHH profile provided by nf-core/configs'
  
  // default BWA
   bwaalnn = 0.04
   bwaalnl = 32
}
 
}
 
// profile specific parameters
profiles {
  pathogen_loose {
    params {
      config_profile_description = 'Pathogen (loose) MPI-SHH profile, provided by nf-core/configs.'
      bwaalnn = 0.01
      bwaalnl = 16
    }
  }
}
 

Tutorial Profiles - Writing your own profile

We will now provide an example of how to write, use and share a project specific profile. We will use the example of Andrades Valtueña et al. 2016.

In it they used the original EAGER (v1) to map DNA from ancient DNA to the genome of the bacterium Yersinia pestis.

Now, we will generate a profile, that, if they were using nf-core/eager they could share with other researchers.

In the methods they described the following:

… reads mapped to Y. pestis CO92 reference with BWA aln (-l 16, -n 0.01, hereby referred to as non-UDG parameters). Reads with mapping quality scores lower than 37 were filtered out. PCR duplicates were removed with MarkDuplicates.”

Furthermore, in their ‘Table 1’ they say they used the NCBI Y. pestis genome ‘NC_003143.1’, which can be found on the NCBI FTP server at: https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/009/065/GCF_000009065.1_ASM906v1/GCF_000009065.1_ASM906v1_genomic.fna.gz

To make a profile with these parameters for use with nf-core/eager we first need to open a text editor, and define a Nextflow ‘profile’ block.

profiles {
 
}
 

Next we need to define the name of the profile. This is what we would write in -profile. Lets call this AndradesValtuena2018.

profiles {
  AndradesValtuena2018 {
 
  }
}

Now we need to make a params ‘scope’. This means these are the parameters you specifically pass to nf-core/eager itself (rather than Nextflow configuration parameters).

You should generally not add non-params scopes in profiles for a specific project. This is because these will normally modify the way the pipeline will run on the computer (rather than just nf-core/eager itself, e.g. the scheduler/executor or maximum memory available), and thus not allow other researchers to reproduce your analysis on their own computer/clusters.

profiles {
  AndradesValtuena2018 {
    params {
 
    }
  }
}

Now, as a cool little trick, we can use a couple of nf-core specific parameters that can help you keep track which profile you are using when running the pipeline. The config_profile_description and config_profile_contact profiles are displayed in the console log when running the pipeline. So you can use these to check if your profile loaded as expected. These are free text boxes so you can put what you like.

profiles {
  AndradesValtuena2018 {
    params {
        config_profile_description = 'non-UDG parameters used in Andrades Valtuena et al. 2018 Curr. Bio.'
        config_profile_contact = 'Aida Andrades Valtueña (@aidaanva)'
    }
  }
}

Now we can add the specific nf-core/eager parameters that will modify the mapping and deduplication parameters in nf-core/eager.

profiles {
  AndradesValtuena2018 {
    params {
        config_profile_description = 'non-UDG parameters used in Andrades Valtuena et al. 2018 Curr. Bio.'
        config_profile_contact = 'Aida Andrades Valtueña (@aidaanva)'
        fasta = 'https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/009/065/GCF_000009065.1_ASM906v1/GCF_000009065.1_ASM906v1_genomic.fna.gz'
        bwaalnn = 0.01
        bwaalnl = 16
        run_bam_filtering = true
        bam_mapping_quality_threshold = 37
        dedupper = 'markduplicates'
    }
  }
}

Once filled in, we can save the file as AndradesValtuena2018.config. This you can use yourself, or upload alongside your publication for others to use.

To use the profile you just need to specify the file containing the profile you wish to use, and then the profile itself.

For example, Aida (Andrades Valtueña) on her cluster sdag at the MPI-SHH (shh) in Jena could run the following:

nextflow run nf-core/eager -c /<path>/<to>/AndradesValtuena2018.config -profile AndradesValtuena2018,sdag,shh --input '/<path>/<to>/<some_input>/' <...>

Then a colleague at a different institution, such as the SciLifeLab, could run the same profile on the UPPMAX cluster in Uppsala with:

nextflow run nf-core/eager -c /<path>/<to>/AndradesValtuena2018.config -profile AndradesValtuena2018,uppmax --input '/<path>/<to>/<some_input>/' <...>

And that’s all there is to it. Of course you should always check that there are no other ‘default’ parameters for your given pipeline are defined in any pipeline-specific or institutional profiles. This ensures that someone re-running the pipeline with your settings is as close to the nf-core/eager defaults as possible, and only settings specific to your given project are used. If there are ‘upstream’ defaults, you should explicitly specify these in your project profile.

Tutorial - How to set up nf-core/eager for human population genetics

Tutorial Human Pop-Gen - Introduction

This tutorial will give a basic example on how to set up nf-core/eager to perform initial screening of samples in the context of ancient human population genetics research.

⚠️ this tutorial does not describe how to install and set up nf-core/eager For this please see other documentation on the nf-co.re website.

We will describe how to set up mapping of ancient sequences against the human reference genome to allow sequencing and library quality-control, estimation of nuclear contamination, genetic sex determination, and production of random draw genotypes in eigenstrat format for a specific set of sites, to be used in further analysis. For this example, I will be using the 1240k SNP set. This SNP set was first described in Mathieson et al. 2015 and contains various positions along the genome that have been extensively genotyped in present-day and ancient populations, and are therefore useful for ancient population genetic analysis. Capture techniques are often used to enrich DNA libraries for fragments, that overlap these SNPs, as is being assumed has been performed in this example.

⚠️ Please be aware that the settings used in this tutorial may not use settings nor produce files you would actually use in ‘real’ analysis. The settings are only specified for demonstration purposes. Please consult the your colleagues, communities and the literature for optimal parameters.

Tutorial Human Pop-Gen - Preparation

Prior setting up the nf-core/eager run, we will need:

1. Raw sequencing data in FASTQ format
2. Reference genome in FASTA format, with associated pre-made bwa, samtools and picard SequenceDictionary indices (however note these can be made for you with nf-core/eager, but this can make a pipeline run take much longer!)
3. A BED file with the positions of the sites of interest.
4. An eigenstrat formatted .snp file for the positions of interest.

We should also ensure we have the very latest version of the nf-core/eager pipeline so we have all latest bugfixes etc. In this case we will be using nf-core/eager version 2.2.0. You should always check on the nf-core website whether a newer release has been made (particularly point releases e.g. 2.2.1).

nextflow pull nf-core/eager -r 2.2.0

It is important to note that if you are planning on running multiple runs of nf-core/eager for a given project, that the version should be kept the same for all runs to ensure consistency in settings for all of your libraries.

Tutorial Human Pop-Gen - Inputs and Outputs

To start, lets make a directory where all your nf-core/eager related files for this run will go, and change into it.

mkdir projectX_preprocessing20200727
cd projectX_preprocessing20200727

The first part of constructing any nf-core/eager run is specifying a few generic parameters that will often be common across all runs. This will be which pipeline, version and profile we will use. We will also specify a unique name of the run to help us keep track of all the nf-core/eager runs you may be running.

nextflow run nf-core/eager \
-r 2.2.0 \
-profile sdag,shh,singularity \
-name 'projectX_preprocessing20200727' \
<...>

For the -profile parameter, I have indicated that I wish to use Singularity as my software container environment, and I will use the MPI-SHH institutional config as listed on nf-core/configs, using the profile for the ‘sdag’ cluster. These profiles specify settings optimised for the specific cluster/institution, such as maximum memory available or which scheduler queues to submit to. More explanations about configs and profiles can be seen in the nf-core website and the profile tutorial.

Next we need to specify our input data. nf-core/eager can accept input FASTQs files in two main ways, either with direct paths to files (with wildcards), or with a Tab-Separate-Value (TSV) file which contains the paths and extra metadata. In this example, we will use the TSV method, as to simulate a realistic use-case, such as receiving paired-end data from an Illumina NextSeq of double-stranded libraries. Illumina NextSeqs sequence a given library across four different ‘lanes’, so for each library you will receive four FASTQ files. The TSV input method is more useful for this context, as it allows ‘merging’ of these lanes after preprocessing prior mapping (whereas direct paths will consider each pair of FASTQ files as independent libraries/samples).

Our TSV file will look something like the following:

Sample_Name     Library_ID      Lane    Colour_Chemistry        SeqType Organism        Strandedness    UDG_Treatment   R1      R2      BAM
EGR001  EGR001.B0101.SG1        1       2       PE      homo_sapiens    double  half    ../../02-raw_data/EGR001.B0101.SG1.1/EGR001.B0101.SG1.1_S0_L001_R1_001.fastq.gz ../../02-raw_data/EGR001.B0101.SG1.1/EGR001.B0101.SG1.1_S0_L001_R2_001.fastq.gz NA
EGR001  EGR001.B0101.SG1        2       2       PE      homo_sapiens    double  half    ../../02-raw_data/EGR001.B0101.SG1.1/EGR001.B0101.SG1.1_S0_L002_R1_001.fastq.gz ../../02-raw_data/EGR001.B0101.SG1.1/EGR001.B0101.SG1.1_S0_L002_R2_001.fastq.gz NA
EGR001  EGR001.B0101.SG1        3       2       PE      homo_sapiens    double  half    ../../02-raw_data/EGR001.B0101.SG1.1/EGR001.B0101.SG1.1_S0_L003_R1_001.fastq.gz ../../02-raw_data/EGR001.B0101.SG1.1/EGR001.B0101.SG1.1_S0_L003_R2_001.fastq.gz NA
EGR001  EGR001.B0101.SG1        4       2       PE      homo_sapiens    double  half    ../../02-raw_data/EGR001.B0101.SG1.1/EGR001.B0101.SG1.1_S0_L004_R1_001.fastq.gz ../../02-raw_data/EGR001.B0101.SG1.1/EGR001.B0101.SG1.1_S0_L004_R2_001.fastq.gz NA
EGR001  EGR001.B0101.SG1        5       2       PE      homo_sapiens    double  half    ../../02-raw_data/EGR001.B0101.SG1.2/EGR001.B0101.SG1.2_S0_L001_R1_001.fastq.gz ../../02-raw_data/EGR001.B0101.SG1.2/EGR001.B0101.SG1.2_S0_L001_R2_001.fastq.gz NA
EGR001  EGR001.B0101.SG1        6       2       PE      homo_sapiens    double  half    ../../02-raw_data/EGR001.B0101.SG1.2/EGR001.B0101.SG1.2_S0_L002_R1_001.fastq.gz ../../02-raw_data/EGR001.B0101.SG1.2/EGR001.B0101.SG1.2_S0_L002_R2_001.fastq.gz NA
EGR001  EGR001.B0101.SG1        7       2       PE      homo_sapiens    double  half    ../../02-raw_data/EGR001.B0101.SG1.2/EGR001.B0101.SG1.2_S0_L003_R1_001.fastq.gz ../../02-raw_data/EGR001.B0101.SG1.2/EGR001.B0101.SG1.2_S0_L003_R2_001.fastq.gz NA
EGR002  EGR002.B0201.SG1        8       2       PE      homo_sapiens    double  half    ../../02-raw_data/EGR001.B0101.SG1.2/EGR001.B0101.SG1.2_S0_L004_R1_001.fastq.gz ../../02-raw_data/EGR001.B0101.SG1.2/EGR001.B0101.SG1.2_S0_L004_R2_001.fastq.gz NA
EGR002  EGR002.B0201.SG1        1       2       PE      homo_sapiens    double  half    ../../02-raw_data/EGR002.B0201.SG1.1/EGR002.B0201.SG1.1_S0_L001_R1_001.fastq.gz ../../02-raw_data/EGR002.B0201.SG1.1/EGR002.B0201.SG1.1_S0_L001_R2_001.fastq.gz NA
EGR002  EGR002.B0201.SG1        2       2       PE      homo_sapiens    double  half    ../../02-raw_data/EGR002.B0201.SG1.1/EGR002.B0201.SG1.1_S0_L002_R1_001.fastq.gz ../../02-raw_data/EGR002.B0201.SG1.1/EGR002.B0201.SG1.1_S0_L002_R2_001.fastq.gz NA
EGR002  EGR002.B0201.SG1        3       2       PE      homo_sapiens    double  half    ../../02-raw_data/EGR002.B0201.SG1.1/EGR002.B0201.SG1.1_S0_L003_R1_001.fastq.gz ../../02-raw_data/EGR002.B0201.SG1.1/EGR002.B0201.SG1.1_S0_L003_R2_001.fastq.gz NA
EGR002  EGR002.B0201.SG1        4       2       PE      homo_sapiens    double  half    ../../02-raw_data/EGR002.B0201.SG1.1/EGR002.B0201.SG1.1_S0_L004_R1_001.fastq.gz ../../02-raw_data/EGR002.B0201.SG1.1/EGR002.B0201.SG1.1_S0_L004_R2_001.fastq.gz NA
EGR002  EGR002.B0201.SG1        5       2       PE      homo_sapiens    double  half    ../../02-raw_data/EGR002.B0201.SG1.2/EGR002.B0201.SG1.2_S0_L001_R1_001.fastq.gz ../../02-raw_data/EGR002.B0201.SG1.2/EGR002.B0201.SG1.2_S0_L001_R2_001.fastq.gz NA
EGR002  EGR002.B0201.SG1        6       2       PE      homo_sapiens    double  half    ../../02-raw_data/EGR002.B0201.SG1.2/EGR002.B0201.SG1.2_S0_L002_R1_001.fastq.gz ../../02-raw_data/EGR002.B0201.SG1.2/EGR002.B0201.SG1.2_S0_L002_R2_001.fastq.gz NA
EGR002  EGR002.B0201.SG1        7       2       PE      homo_sapiens    double  half    ../../02-raw_data/EGR002.B0201.SG1.2/EGR002.B0201.SG1.2_S0_L003_R1_001.fastq.gz ../../02-raw_data/EGR002.B0201.SG1.2/EGR002.B0201.SG1.2_S0_L003_R2_001.fastq.gz NA
EGR002  EGR002.B0201.SG1        8       2       PE      homo_sapiens    double  half    ../../02-raw_data/EGR002.B0201.SG1.2/EGR002.B0201.SG1.2_S0_L004_R1_001.fastq.gz ../../02-raw_data/EGR002.B0201.SG1.2/EGR002.B0201.SG1.2_S0_L004_R2_001.fastq.gz NA

You can see that we have a single line for each pair of FASTQ files representing each Lane, but the Sample_Name and Library_ID columns identify and group them together accordingly. Secondly, as we have NextSeq data, we have specified we have 2 for Colour_Chemistry, which is important for downstream processing (see below). See the nf-core/eager parameter documentation above for more specifications on how to set up a TSV file (e.g. why despite NextSeqs only having 4 lanes, we go up to 8 in the example above).

Alongside our input TSV file, we will also specify the paths to our reference FASTA file and the corresponding indices.

nextflow run nf-core/eager \
-r 2.2.0 \
-profile sdag,shh,singularity \
-name 'projectX_preprocessing20200727' \
--input 'preprocessing20200727.tsv' \
--fasta '../Reference/genome/hs37d5.fa' \
--bwa_index '../Reference/genome/hs37d5/' \
--fasta_index '../Reference/genome/hs37d5.fa.fai' \
--seq_dict '../Reference/genome/hs37d5.dict' \
<...>

We specify the paths to each reference genome and it’s corresponding tool specific index. Paths should always be encapsulated in quotes to ensure Nextflow evaluates them, rather than your shell! Also note that as bwa generates multiple index files, nf-core/eager takes a directory that must contain these indices instead.

Note the difference between single and double - parameters. The former represent Nextflow flags, while the latter are nf-core/eager specific flags.

Finally, we can also specify the output directory and the Nextflow work/ directory (which contains ‘intermediate’ working files and directories).

nextflow run nf-core/eager \
-r 2.2.0 \
-profile sdag,shh,singularity \
-name 'projectX_preprocessing20200727' \
--input 'preprocessing20200727.tsv' \`
--fasta '../Reference/genome/hs37d5.fa' \
--bwa_index '../Reference/genome/hs37d5/' \
--fasta_index '../Reference/genome/hs37d5.fa.fai' \
--seq_dict '../Reference/genome/hs37d5.dict' \
--outdir './results/' \
- w './work/' \
<...>

Tutorial Human Pop-Gen - Pipeline Configuration

Now that we have specified the input data, we can start moving onto specifying settings for each different module we will be running. As mentioned above, we are pretending to run with NextSeq data, which is generated with a two-colour imaging technique. What this means is when you have shorter molecules than the number of cycles of the sequencing chemistry, the sequencer will repeatedly see ‘G’ calls (no colour) at the last few cycles, and you get long poly-G ‘tails’ on your reads. We therefore will turn on the poly-G clipping functionality offered by fastp, and any pairs of files indicated in the TSV file as having 2 in the Colour_Chemistry column will be passed to fastp. We will not change the default minimum length of a poly-G string to be clipped.

nextflow run nf-core/eager \
-r 2.2.0 \
-profile sdag,shh,singularity \
-name 'projectX_preprocessing20200727' \
--input 'preprocessing20200727.tsv' \
--fasta '../Reference/genome/hs37d5.fa' \
--bwa_index '../Reference/genome/hs37d5/' \
--fasta_index '../Reference/genome/hs37d5.fa.fai' \
--seq_dict '../Reference/genome/hs37d5.dict' \
--outdir './results/' \
- w './work/' \
--complexity_filter_poly_g \
<...>

Since our input data is paired-end, we will be using DeDup for duplicate removal, which takes into account both the start and end of a merged read before flagging it as a duplicate. To ensure this happens works properly we first need to disable base quality trimming of collapsed reads within Adapter Removal. To do this, we will provide the option --preserve5p. Additionally, Dedup should only be provided with merged reads, so we will need to provide the option --mergedonly here as well. We can then specify which dedupper we want to use with --dedupper.

nextflow run nf-core/eager \
-r 2.2.0 \
-profile sdag,shh,singularity \
-name 'projectX_preprocessing20200727' \
--input 'preprocessing20200727.tsv' \
--fasta '../Reference/genome/hs37d5.fa' \
--bwa_index '../Reference/genome/hs37d5/' \
--fasta_index '../Reference/genome/hs37d5.fa.fai' \
--seq_dict '../Reference/genome/hs37d5.dict' \
--outdir './results/' \
- w './work/' \
--complexity_filter_poly_g \
--preserve5p \
--mergedonly \
--dedupper 'dedup' \
<...>

We then need to specify the mapping parameters for this run. The default mapping parameters of nf-core/eager are fine for the purposes of our run. Personally, I like to set --bwaalnn to 0.01, (down from the default 0.04) which reduces the stringency in the number of allowed mismatches between the aligned sequences and the reference.

nextflow run nf-core/eager \
-r 2.2.0 \
-profile sdag,shh,singularity \
-name 'projectX_preprocessing20200727' \
--input 'preprocessing20200727.tsv' \
--fasta '../Reference/genome/hs37d5.fa' \
--bwa_index '../Reference/genome/hs37d5/' \
--fasta_index '../Reference/genome/hs37d5.fa.fai' \
--seq_dict '../Reference/genome/hs37d5.dict' \
--outdir './results/' \
- w './work/' \
--complexity_filter_poly_g \
--preserve5p \
--mergedonly \
--dedupper 'dedup' \
--bwaalnn 0.01 \
<...>

We may also want to remove ambiguous sequences from our alignments, and also remove off-target reads to speed up downstream processing (and reduce your hard-disk footprint). We can do this with the samtools filter module to set a mapping-quality filter (e.g. with a value of 25 to retain only slightly ambiguous alignments that might occur from damage), and to indicate to discard unmapped reads.

nextflow run nf-core/eager \
-r 2.2.0 \
-profile sdag,shh,singularity \
-name 'projectX_preprocessing20200727' \
--input 'preprocessing20200727.tsv' \
--fasta '../Reference/genome/hs37d5.fa' \
--bwa_index '../Reference/genome/hs37d5/' \
--fasta_index '../Reference/genome/hs37d5.fa.fai' \
--seq_dict '../Reference/genome/hs37d5.dict' \
--outdir './results/' \
- w './work/' \
--complexity_filter_poly_g \
--preserve5p \
--mergedonly \
--dedupper 'dedup' \
--bwaalnn 0.01 \
--run_bam_filtering \
--bam_mapping_quality_threshold 25 \
--bam_unmapped_type 'discard' \
<...>

Next, we will set up trimming of the mapped reads to alleviate the effects of DNA damage during genotyping. To do this we will activate trimming with --run_trim_bam. The libraries in this underwent ‘half’ UDG treatment. This will generally restrict all remaining DNA damage to the first 2 base pairs of a fragment. We will therefore use --bamutils_clip_half_udg_left and --bamutils_clip_half_udg_right to trim 2bp on either side of each fragment.

nextflow run nf-core/eager \
-r 2.2.0 \
-profile sdag,shh,singularity \
-name 'projectX_preprocessing20200727' \
--input 'preprocessing20200727.tsv' \
--fasta '../Reference/genome/hs37d5.fa' \
--bwa_index '../Reference/genome/hs37d5/' \
--fasta_index '../Reference/genome/hs37d5.fa.fai' \
--seq_dict '../Reference/genome/hs37d5.dict' \
--outdir './results/' \
- w './work/' \
--complexity_filter_poly_g \
--preserve5p \
--mergedonly \
--dedupper 'dedup' \
--bwaalnn 0.01 \
--run_bam_filtering \
--bam_mapping_quality_threshold 25 \
--bam_unmapped_type 'discard' \
--run_trim_bam \
--bamutils_clip_half_udg_left 2 \
--bamutils_clip_half_udg_right 2 \
<...>

To activate human sex determination (using Sex.DetERRmine.py) we will provide the option --run_sexdeterrmine. Additionally, we will provide sexdeterrmine with the BED file of our SNPs of interest using the --sexdeterrmine_bedfile flag. Here I will use the 1240k SNP set as an example. This will cut down on computational time and also providing us with an error bar around the relative coverage on the X and Y chromosomes.

nextflow run nf-core/eager \
-r 2.2.0 \
-profile sdag,shh,singularity \
-name 'projectX_preprocessing20200727' \
--input 'preprocessing20200727.tsv' \
--fasta '../Reference/genome/hs37d5.fa' \
--bwa_index '../Reference/genome/hs37d5/' \
--fasta_index '../Reference/genome/hs37d5.fa.fai' \
--seq_dict '../Reference/genome/hs37d5.dict' \
--outdir './results/' \
- w './work/' \
--complexity_filter_poly_g \
--preserve5p \
--mergedonly \
--dedupper 'dedup' \
--bwaalnn 0.01 \
--run_bam_filtering \
--bam_mapping_quality_threshold 25 \
--bam_unmapped_type 'discard' \
--run_trim_bam \
--bamutils_clip_half_udg_left 2 \
--bamutils_clip_half_udg_right 2 \
--run_sexdeterrmine \
--sexdeterrmine_bedfile '../Reference/genome/1240k.sites.bed' \
<...>

Similarly, we will activate nuclear contamination estimation with --run_nuclear_contamination. This process requires us to also specify the contig name of the X chromosome in the reference genome we are using with --contamination_chrom_name. Here, we are using hs37d5, where the X chromosome is simply named ‘X’.

nextflow run nf-core/eager \
-r 2.2.0 \
-profile sdag,shh,singularity \
-name 'projectX_preprocessing20200727' \
--input 'preprocessing20200727.tsv' \
--fasta '../Reference/genome/hs37d5.fa' \
--bwa_index '../Reference/genome/hs37d5/' \
--fasta_index '../Reference/genome/hs37d5.fa.fai' \
--seq_dict '../Reference/genome/hs37d5.dict' \
--outdir './results/' \
- w './work/' \
--complexity_filter_poly_g \
--preserve5p \
--mergedonly \
--dedupper 'dedup' \
--bwaalnn 0.01 \
--run_bam_filtering \
--bam_mapping_quality_threshold 25 \
--bam_unmapped_type 'discard' \
--run_trim_bam \
--bamutils_clip_half_udg_left 2 \
--bamutils_clip_half_udg_right 2 \
--run_sexdeterrmine \
--sexdeterrmine_bedfile '../Reference/genome/1240k.sites.bed' \
--run_nuclear_contamination \
--contamination_chrom_name 'X' \
<...>

Because nuclear contamination estimates can only be provided for males, it is possible that we will need to get mitochondrial DNA contamination estimates for any females in our dataset. This cannot be done within nf-core/eager (v2.2.0) and we will need to do this manually at a later time. However, mtDNA contamination estimates have been shown to only be reliable for nuclear contamination when the ratio of mitochondrial to nuclear reads is low (Furtwängler et al. 2018). We can have nf-core/eager calculate that ratio for us with --run_mtnucratio, and providing the name of the mitochondrial DNA contig in our reference genome with --mtnucratio_header. Within hs37d5, the mitochondrial contig is named ‘MT’.

nextflow run nf-core/eager \
-r 2.2.0 \
-profile sdag,shh,singularity \
-name 'projectX_preprocessing20200727' \
--input 'preprocessing20200727.tsv' \
--fasta '../Reference/genome/hs37d5.fa' \
--bwa_index '../Reference/genome/hs37d5/' \
--fasta_index '../Reference/genome/hs37d5.fa.fai' \
--seq_dict '../Reference/genome/hs37d5.dict' \
--outdir './results/' \
- w './work/' \
--complexity_filter_poly_g \
--preserve5p \
--mergedonly \
--dedupper 'dedup' \
--bwaalnn 0.01 \
--run_bam_filtering \
--bam_mapping_quality_threshold 25 \
--bam_unmapped_type 'discard' \
--run_trim_bam \
--bamutils_clip_half_udg_left 2 \
--bamutils_clip_half_udg_right 2 \
--run_sexdeterrmine \
--sexdeterrmine_bedfile '../Reference/genome/1240k.sites.bed' \
--run_nuclear_contamination \
--contamination_chrom_name 'X' \
--run_mtnucratio \
--mtnucratio_header 'MT' \
<...>

Finally, we need to specify genotyping parameters. First, we need to activate genotyping with --run_genotyping. It is also important to specify we wish to use the trimmed data for genotyping, to avoid the effects of DNA damage. To do this, we will specify the --genotyping_source as 'trimmed'. Then we can specify the genotyping tool to use with --genotyping_tool. We will be using 'pileupCaller' to produce random draw genotypes in eigenstrat format. For this process we will need to specify a BED file of the sites of interest (the same as before) with --pileupcaller_bedfile, as well as an eigenstrat formatted .snp file of these sites that is specified with --pileupcaller_snpfile.

nextflow run nf-core/eager \
-r 2.2.0 \
-profile sdag,shh,singularity \
-name 'projectX_preprocessing20200727' \
--input 'preprocessing20200727.tsv' \
--fasta '../Reference/genome/hs37d5.fa' \
--bwa_index '../Reference/genome/hs37d5/' \
--fasta_index '../Reference/genome/hs37d5.fa.fai' \
--seq_dict '../Reference/genome/hs37d5.dict' \
--outdir './results/' \
- w './work/' \
--complexity_filter_poly_g \
--preserve5p \
--mergedonly \
--dedupper 'dedup' \
--bwaalnn 0.01 \
--run_bam_filtering \
--bam_mapping_quality_threshold 25 \
--bam_unmapped_type 'discard' \
--run_trim_bam \
--bamutils_clip_half_udg_left 2 \
--bamutils_clip_half_udg_right 2 \
--run_sexdeterrmine \
--sexdeterrmine_bedfile '../Reference/genome/1240k.sites.bed' \
--run_nuclear_contamination \
--contamination_chrom_name 'X' \
--run_mtnucratio \
--mtnucratio_header 'MT' \
--run_genotyping \
--genotyping_source 'trimmed' \
--genotyping_tool 'pileupcaller' \
--pileupcaller_bedfile '../Reference/genome/1240k.sites.bed' \
--pileupcaller_snpfile '../Datasets/1240k/1240k.snp'

With this, we are ready to submit! If running on a remote cluster/server, Make sure to run this in a screen session or similar, so that if you get a ssh signal drop or want to log off, Nextflow will not crash.

Tutorial Human Pop-Gen - Results

Assuming the run completed without any crashes (if problems do occur, check against #usage that all parameters are as expected, or check the FAQ), we can now check our results in results/.

Tutorial Human Pop-Gen - MultiQC Report

In here there are many different directories containing different output files. The first directory to check is the MultiQC/ directory. You should find a multiqc_report.html file. You will need to view this in a web browser, so I recommend either mounting your server to your file browser, or downloading it to your own local machine (PC/Laptop etc.).

Once you’ve opened this you can go through each section and evaluate all the results. You will likely want to check these for artefacts (e.g. weird damage patterns on the human DNA, or weirdly skewed coverage distributions).

For example, I normally look for things like:

General Stats Table:

• Do I see the expected number of raw sequencing reads (summed across each set of FASTQ files per library) that was requested for sequencing?
• Does the percentage of trimmed reads look normal for aDNA, and do lengths after trimming look short as expected of aDNA?
• Does ClusterFactor or ‘Dups’ look high (e.g. >2 or >10% respectively) suggesting over-amplified or badly preserved samples?
• Do the mapped reads show increased frequency of C>Ts on the 5’ end of molecules?
• Is the number of SNPs used for nuclear contamination really low for any individuals (e.g. < 100)? If so, then the estimates might not be very accurate.

FastQC (pre-AdapterRemoval):

• Do I see any very early drop off of sequence quality scores suggesting a problematic sequencing run?
• Do I see outlier GC content distributions?
• Do I see high sequence duplication levels?

AdapterRemoval:

• Do I see high numbers of singletons or discarded read pairs?

FastQC (post-AdapterRemoval):

• Do I see improved sequence quality scores along the length of reads?
• Do I see reduced adapter content levels?

Samtools Flagstat (pre/post Filter):

• Do I see outliers, e.g. with unusually high levels of human DNA, (indicative of contamination) that require downstream closer assessment? Are your samples exceptionally preserved? If not, a value higher than e.g. 50% might require your attention.

DeDup/Picard MarkDuplicates:

• Do I see large numbers of duplicates being removed, possibly indicating over-amplified or badly preserved samples?

DamageProfiler:

• Do I see evidence of damage on human DNA?
  • High numbers of mapped reads but no damage may indicate significant modern contamination.
  • Was the read trimming I specified enough to overcome damage effects?

SexDetERRmine:

• Do the relative coverages on the X and Y chromosome fall within the expected areas of the plot?
• Do all individuals have enough data for accurate sex determination?
• Do the proportions of autosomal/X/Y reads make sense? If there is an overrepresentation of reads within one bin, is the data enriched for that bin?

Detailed documentation and descriptions for all MultiQC modules can be seen in the the ‘Documentation’ folder of the results directory or here in the output documentation

If you’re happy everything looks good in terms of sequencing, we then look at specific directories to find any files you might want to use for downstream processing.

Note that when you get back to writing up your publication, all the versions of the tools can be found under the ‘nf-core/eager Software Versions’ section of the MultiQC report. But be careful! All tools in the container are listed, so you may have to remove some of them that you didn’t actually use in the set up.

For example, in this example, we have used: Nextflow, nf-core/eager, FastQC, AdapterRemoval, fastP, BWA, Samtools, endorS.py, DeDup, Qualimap, PreSeq, DamageProfiler, bamUtil, sexdeterrmine, angsd, MTNucRatioCalculator, sequenceTools, and MultiQC.

Citations to all used tools can be seen here

Tutorial Human Pop-Gen - Files for Downstream Analysis

You will find the eigenstrat dataset containing the random draw genotypes of your run in the genotyping/ directory. Genotypes from double stranded libraries, like the ones in this example, are found in the dataset pileupcaller.double.{geno,snp,ind}.txt, while genotypes for any single stranded libraries will instead be in pileupcaller.single.{geno,snp,ind}.txt.

Tutorial Human Pop-Gen - Clean up

Finally, I would recommend cleaning up your work/ directory of any intermediate files (if your -profile does not already do so). You can do this by going to above your results/ and work/ directory, e.g.

cd /<path>/<to>/projectX_preprocessing20200727

and running

nextflow clean -f -k

Tutorial Human Pop-Gen - Summary

In this this tutorial we have described an example on how to set up an nf-core/eager run to preprocess human aDNA for population genetic studies, preform some simple quality control checks, and generate random draw genotypes for downstream analysis of the data. Additionally, we described what to look for in the run summary report generated by MultiQC and where to find output files that can be used for downstream analysis.

Tutorial - How to set up nf-core/eager for metagenomic screening

Tutorial Metagenomics - Introduction

The field of archaeogenetics is now expanding out from analysing the genomes of single organisms but to whole communities of taxa. One particular example is of human associated microbiomes, as preserved in ancient palaeofaeces (gut) or dental calculus (oral). This tutorial will give a basic example on how to set up nf-core/eager to perform initial screening of samples in the context of ancient microbiome research.

⚠️ this tutorial does not describe how to install and set up nf-core/eager For this please see other documentation on the nf-co.re website.

We will describe how to set up mapping of ancient dental calculus samples against the human reference genome to allow sequencing and library quality-control, but additionally perform taxonomic profiling of the off-target reads from this mapping using MALT, and perform aDNA authentication with HOPS.

⚠️ Please be aware that the settings used in this tutorial may not use settings nor produce files you would actually use in ‘real’ analysis. The settings are only specified for demonstration purposes. Please consult the your colleagues, communities and the literature for optimal parameters.

Tutorial Metagenomics - Preparation

Prior setting up an nf-core/eager run for metagenomic screening, we will need:

1. Raw sequencing data in FASTQ format
2. Reference genome in FASTA format, with associated pre-made bwa, samtools and picard SequenceDictionary indices
3. A MALT database of your choice (see MALT manual for set-up)
4. A list of (NCBI) taxa containing well-known taxa of your microbiome (see below)
5. HOPS resources .map and .tre files (available here)

We should also ensure we have the very latest version of the nf-core/eager pipeline so we have all latest bugfixes etc. In this case we will be using nf-core/eager version 2.2.0. You should always check on the nf-core website whether a newer release has been made (particularly point releases e.g. 2.2.1).

nextflow pull nf-core/eager -r 2.2.0

It is important to note that if you are planning on running multiple runs of nf-core/eager for a given project, that the version should be kept the same for all runs to ensure consistency in settings for all of your libraries.

Tutorial Metagenomics - Inputs and Outputs

To start, lets make a directory where all your nf-core/eager related files for this run will go, and change into it.

mkdir projectX_screening20200720
cd projectX_screening20200720

The first part of constructing any nf-core/eager run is specifying a few generic parameters that will often be common across all runs. This will be which pipeline, version and profile we will use. We will also specify a unique name of the run to help us keep track of all the nf-core/eager runs you may be running.

nextflow run nf-core/eager \
-r 2.2.0 \
-profile sdag,shh,singularity \
-name 'projectX_screening20200720' \
<...>

For the -profile parameter, I have indicated that I wish to use Singularity as my software container environment, and I will use the MPI-SHH institutional config as listed on nf-core/configs, and using the profile for the ‘sdag’ cluster. These profiles specify settings optimised for the specific cluster/institution, such as maximum memory available or which scheduler queues to submit to. More explanations about configs and profiles can be seen in the nf-core website and the profile tutorial.

Next we need to specify our input data. nf-core/eager can accept input FASTQs files in two main ways, either with direct paths to files (with wildcards), or with a Tab-Separate-Value (TSV) file which contains the paths and extra metadata. In this example, we will use the TSV method, as to simulate a realistic use-case, such as receiving paired-end data from an Illumina NextSeq of double-stranded libraries. Illumina NextSeqs sequence a given library across four different ‘lanes’, so for each library you will receive four FASTQ files. The TSV input method is more useful for this context, as it allows ‘merging’ of these lanes after preprocessing prior mapping (whereas direct paths will consider each pair of FASTQ files as independent libraries/samples).

Our TSV file will look something like the following:

Sample_Name     Library_ID      Lane    Colour_Chemistry        SeqType Organism        Strandedness    UDG_Treatment   R1      R2      BAM
EGR001  EGR001.B0101.SG1        1       2       PE      homo_sapiens    double  half    ../../02-raw_data/EGR001.B0101.SG1.1/EGR001.B0101.SG1.1_S0_L001_R1_001.fastq.gz ../../02-raw_data/EGR001.B0101.SG1.1/EGR001.B0101.SG1.1_S0_L001_R2_001.fastq.gz NA
EGR001  EGR001.B0101.SG1        2       2       PE      homo_sapiens    double  half    ../../02-raw_data/EGR001.B0101.SG1.1/EGR001.B0101.SG1.1_S0_L002_R1_001.fastq.gz ../../02-raw_data/EGR001.B0101.SG1.1/EGR001.B0101.SG1.1_S0_L002_R2_001.fastq.gz NA
EGR001  EGR001.B0101.SG1        3       2       PE      homo_sapiens    double  half    ../../02-raw_data/EGR001.B0101.SG1.1/EGR001.B0101.SG1.1_S0_L003_R1_001.fastq.gz ../../02-raw_data/EGR001.B0101.SG1.1/EGR001.B0101.SG1.1_S0_L003_R2_001.fastq.gz NA
EGR001  EGR001.B0101.SG1        4       2       PE      homo_sapiens    double  half    ../../02-raw_data/EGR001.B0101.SG1.1/EGR001.B0101.SG1.1_S0_L004_R1_001.fastq.gz ../../02-raw_data/EGR001.B0101.SG1.1/EGR001.B0101.SG1.1_S0_L004_R2_001.fastq.gz NA
EGR001  EGR001.B0101.SG1        5       2       PE      homo_sapiens    double  half    ../../02-raw_data/EGR001.B0101.SG1.2/EGR001.B0101.SG1.2_S0_L001_R1_001.fastq.gz ../../02-raw_data/EGR001.B0101.SG1.2/EGR001.B0101.SG1.2_S0_L001_R2_001.fastq.gz NA
EGR001  EGR001.B0101.SG1        6       2       PE      homo_sapiens    double  half    ../../02-raw_data/EGR001.B0101.SG1.2/EGR001.B0101.SG1.2_S0_L002_R1_001.fastq.gz ../../02-raw_data/EGR001.B0101.SG1.2/EGR001.B0101.SG1.2_S0_L002_R2_001.fastq.gz NA
EGR001  EGR001.B0101.SG1        7       2       PE      homo_sapiens    double  half    ../../02-raw_data/EGR001.B0101.SG1.2/EGR001.B0101.SG1.2_S0_L003_R1_001.fastq.gz ../../02-raw_data/EGR001.B0101.SG1.2/EGR001.B0101.SG1.2_S0_L003_R2_001.fastq.gz NA
EGR002  EGR002.B0201.SG1        8       2       PE      homo_sapiens    double  half    ../../02-raw_data/EGR001.B0101.SG1.2/EGR001.B0101.SG1.2_S0_L004_R1_001.fastq.gz ../../02-raw_data/EGR001.B0101.SG1.2/EGR001.B0101.SG1.2_S0_L004_R2_001.fastq.gz NA
EGR002  EGR002.B0201.SG1        1       2       PE      homo_sapiens    double  half    ../../02-raw_data/EGR002.B0201.SG1.1/EGR002.B0201.SG1.1_S0_L001_R1_001.fastq.gz ../../02-raw_data/EGR002.B0201.SG1.1/EGR002.B0201.SG1.1_S0_L001_R2_001.fastq.gz NA
EGR002  EGR002.B0201.SG1        2       2       PE      homo_sapiens    double  half    ../../02-raw_data/EGR002.B0201.SG1.1/EGR002.B0201.SG1.1_S0_L002_R1_001.fastq.gz ../../02-raw_data/EGR002.B0201.SG1.1/EGR002.B0201.SG1.1_S0_L002_R2_001.fastq.gz NA
EGR002  EGR002.B0201.SG1        3       2       PE      homo_sapiens    double  half    ../../02-raw_data/EGR002.B0201.SG1.1/EGR002.B0201.SG1.1_S0_L003_R1_001.fastq.gz ../../02-raw_data/EGR002.B0201.SG1.1/EGR002.B0201.SG1.1_S0_L003_R2_001.fastq.gz NA
EGR002  EGR002.B0201.SG1        4       2       PE      homo_sapiens    double  half    ../../02-raw_data/EGR002.B0201.SG1.1/EGR002.B0201.SG1.1_S0_L004_R1_001.fastq.gz ../../02-raw_data/EGR002.B0201.SG1.1/EGR002.B0201.SG1.1_S0_L004_R2_001.fastq.gz NA
EGR002  EGR002.B0201.SG1        5       2       PE      homo_sapiens    double  half    ../../02-raw_data/EGR002.B0201.SG1.2/EGR002.B0201.SG1.2_S0_L001_R1_001.fastq.gz ../../02-raw_data/EGR002.B0201.SG1.2/EGR002.B0201.SG1.2_S0_L001_R2_001.fastq.gz NA
EGR002  EGR002.B0201.SG1        6       2       PE      homo_sapiens    double  half    ../../02-raw_data/EGR002.B0201.SG1.2/EGR002.B0201.SG1.2_S0_L002_R1_001.fastq.gz ../../02-raw_data/EGR002.B0201.SG1.2/EGR002.B0201.SG1.2_S0_L002_R2_001.fastq.gz NA
EGR002  EGR002.B0201.SG1        7       2       PE      homo_sapiens    double  half    ../../02-raw_data/EGR002.B0201.SG1.2/EGR002.B0201.SG1.2_S0_L003_R1_001.fastq.gz ../../02-raw_data/EGR002.B0201.SG1.2/EGR002.B0201.SG1.2_S0_L003_R2_001.fastq.gz NA
EGR002  EGR002.B0201.SG1        8       2       PE      homo_sapiens    double  half    ../../02-raw_data/EGR002.B0201.SG1.2/EGR002.B0201.SG1.2_S0_L004_R1_001.fastq.gz ../../02-raw_data/EGR002.B0201.SG1.2/EGR002.B0201.SG1.2_S0_L004_R2_001.fastq.gz NA

You can see that we have a single line for each pair of FASTQ files representing each Lane, but the Sample_Name and Library_ID columns identify and group them together accordingly. Secondly, as we have NextSeq data, we have specified we have 2 for Colour_Chemistry, which is important for downstream processing (see below). The other columns are less important for this particular context of metagenomic screening. See the nf-core/eager usage documentation for more specifications on how to set up a TSV file (e.g. why despite NextSeqs only having 4 lanes, we go up to 8 in the example above).

Alongside our input TSV file, we will also specify the paths to our reference FASTA file and the corresponding indices.

nextflow run nf-core/eager \
-r 2.2.0 \
-profile sdag,shh,singularity \
-name 'projectX_screening20200720' \
--input 'screening20200720.tsv' \
--fasta '../Reference/genome/GRCh38.fa' \
--bwa_index '../Reference/genome/GRCh38/' \
--fasta_index '../Reference/genome/GRCh38.fa.fai' \
--seq_dict '../Reference/genome/GRCh38.dict' \
<...>

We specify the paths to each reference genome and it’s corresponding tool specific index. Paths should always be encapsulated in quotes to ensure Nextflow evaluates them, rather than your shell! Also note that as bwa generates multiple index files, nf-core/eager takes a directory that must contain these indices instead.

Note the difference between single and double - parameters. The former represent Nextflow flags, while double are nf-core/eager specific flags.

Finally, we can also specify the output directory and the Nextflow work/ directory (which contains ‘intermediate’ working files and directories).

nextflow run nf-core/eager \
-r 2.2.0 \
-profile sdag,shh,singularity \
-name 'projectX_screening20200720' \
--input 'screening20200720.tsv' \
--fasta '../Reference/genome/GRCh38.fa' \
--bwa_index '../Reference/genome/GRCh38/' \
--fasta_index '../Reference/genome/GRCh38.fa.fai' \
--seq_dict '../Reference/genome/GRCh38.dict' \
--outdir './results/' \
- w './work/' \
<...>

Tutorial Metagenomics - Pipeline Configuration

Now that we have specified the input data, we can start moving onto specifying settings for each different module we will be running. As mentioned above, we are pretending to run with NextSeq data, which is generated with a two-colour imaging technique. What this means is when you have shorter molecules than the number of cycles of the sequencing chemistry, the sequencer will repeatedly see ‘G’ calls (no colour) at the last few cycles, and you get long poly-G ‘tails’ on your reads. We therefore will turn on the poly-G clipping functionality offered by fastp, and any pairs of files indicated in the TSV file as having 2 in the Colour_Chemistry column will be passed to fastp. We will not change the default minimum length of a poly-G string to be clipped.

nextflow run nf-core/eager \
-r 2.2.0 \
-profile sdag,shh,singularity \
-name 'projectX_screening20200720' \
--input 'screening20200720.tsv' \
--fasta '../Reference/genome/GRCh38.fa' \
--bwa_index '../Reference/genome/GRCh38/' \
--fasta_index '../Reference/genome/GRCh38.fa.fai' \
--seq_dict '../Reference/genome/GRCh38.dict' \
--outdir './results/' \
- w './work/' \
--complexity_filter_poly_g \
<...>

We will keep the default settings for mapping etc. against the reference genome as we will only use this for sequencing quality control, however we now need to specify that we want to run metagenomic screening. To do this we firstly need to tell nf-core/eager what to do with the off target reads from the mapping.

nextflow run nf-core/eager \
-r 2.2.0 \
-profile sdag,shh,singularity \
-name 'projectX_screening20200720' \
--input 'screening20200720.tsv' \
--fasta '../Reference/genome/GRCh38.fa' \
--bwa_index '../Reference/genome/GRCh38/' \
--fasta_index '../Reference/genome/GRCh38.fa.fai' \
--seq_dict '../Reference/genome/GRCh38.dict' \
--outdir './results/' \
- w './work/' \
--complexity_filter_poly_g \
--run_bam_filtering \
--bam_unmapped_type 'fastq' \
<...>