nf-core/nanoseq

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Introduction

You will need to create a file with information about the samples in your experiment/run before executing the pipeline. Use the --input parameter to specify its location. It has to be a comma-separated file with 5 columns and a header row:

Specifying a reference genome/transcriptome

Each sample in the sample sheet can be mapped to its own reference genome or transcriptome. Please see below for additional details required to fill in the genome and transcriptome columns appropriately:

• If both genome and transcriptome are not specified then the mapping will be skipped for that sample.
• If both genome and transcriptome are specified as local fasta files then the transcriptome will be preferentially used for mapping.
• If genome is specified as a local fasta file and transcriptome is left blank then mapping will be performed relative to the genome.
• If genome isnt specified and transcriptome is provided as a fasta file then mapping will be performed relative to the transcriptome.
• If genome is specified as an AWS iGenomes key then the transcriptome column can be blank. The associated gtf file for the transcriptome will be automatically obtained in order to create a transcriptome fasta file. However, the reads will only be mapped to the transcriptome if --protocol cDNA or --protocol directRNA. If --protocol DNA then the reads will still be mapped to the genome essentially ignoring the gtf file.
• If genome is specified as a local fasta file and transcriptome is a specified as a local gtf file then both of these will be used to create a transcriptome fasta file. However, the reads will only be mapped to the transcriptome if --protocol cDNA or --protocol directRNA. If --protocol DNA then the reads will still be mapped to the genome essentially ignoring the gtf file.

Skip basecalling/demultiplexing

As shown in the examples below, the accepted format of the file is slightly different if you would like to run the pipeline with or without basecalling/demultiplexing.

With basecalling and demultiplexing

Example samplesheet.csv for barcoded fast5 inputs

group,replicate,barcode,input_file,genome,transcriptome
WT_MOUSE,1,1,,mm10,
WT_HUMAN,1,2,,hg19,
WT_POMBE,1,3,,/path/to/local/genome.fa,
WT_DENOVO,1,4,,,/path/to/local/transcriptome.fa
WT_LOCAL,2,5,,/path/to/local/genome.fa,/path/to/local/transcriptome.gtf
WT_UNKNOWN,3,6,,,

Example command for barcoded fast5 inputs

nextflow run nf-core/nanoseq \
    --input samplesheet.csv \
    --protocol cDNA \
    --input_path ./fast5/ \
    --flowcell FLO-MIN106 \
    --kit SQK-DCS109 \
    --barcode_kit EXP-NBD103 \
    -profile <docker/singularity/institute>

With basecalling but not demultiplexing

Example samplesheet.csv for non-barcoded fast5 inputs

group,replicate,barcode,input_file,genome,transcriptome
SAMPLE,1,1,/path/to/local/genome.fa,,

Only a single sample can be specified if you would like to skip demultiplexing

Example command for non-barcoded fast5 inputs

nextflow run nf-core/nanoseq \
    --input samplesheet.csv \
    --protocol cDNA \
    --input_path ./fast5/ \
    --flowcell FLO-MIN106 \
    --kit SQK-DCS108 \
    --skip_demultiplexing \
    -profile <docker/singularity/institute>

With demultiplexing but not basecalling

Example samplesheet.csv for non-demultiplexed fastq inputs

group,replicate,barcode,input_file,genome,transcriptome
WT_MOUSE,1,1,,mm10,
WT_HUMAN,1,2,,hg19,
WT_POMBE,1,3,,/path/to/local/genome.fa,
WT_DENOVO,1,4,,,/path/to/local/transcriptome.fa
WT_LOCAL,2,5,,/path/to/local/genome.fa,/path/to/local/transcriptome.gtf
WT_UNKNOWN,3,6,,,

Example command for non-demultiplexed fastq inputs

nextflow run nf-core/nanoseq \
    --input samplesheet.csv \
    --protocol DNA \
    --input_path ./undemultiplexed.fastq.gz \
    --barcode_kit 'NBD103/NBD104' \
    --skip_basecalling \
    -profile <docker/singularity/institute>

Without both basecalling and demultiplexing

Example samplesheet.csv for demultiplexed fastq inputs

group,replicate,barcode,input_file,genome,transcriptome
WT,1,,SAM101A1.fastq.gz,hg19,
WT,2,,SAM101A2.fastq.gz,hg19,
KO,1,,SAM101A3.fastq.gz,hg19,
KO,2,,SAM101A4.fastq.gz,hg19,

Example command for demultiplexed fastq inputs

nextflow run nf-core/nanoseq \
    --input samplesheet.csv \
    --protocol cDNA \
    --skip_basecalling \
    --skip_demultiplexing \
    -profile <docker/singularity/institute>

Without basecalling, demultiplexing, and alignment

Example samplesheet.csv for BAM inputs

group,replicate,barcode,input_file,genome,transcriptome
WT,1,,SAM101A1.bam,hg19,
WT,2,,SAM101A2.bam,hg19,
KO,1,,SAM101A3.bam,hg19,
KO,2,,SAM101A4.bam,hg19,

Example command for BAM inputs

nextflow run nf-core/nanoseq \
    --input samplesheet.csv \
    --protocol cDNA \
    --skip_basecalling \
    --skip_demultiplexing \
    --skip_alignment \
    -profile <docker/singularity/institute>

Running the pipeline

The typical command for running the pipeline is as follows:

nextflow run nf-core/nanoseq \
    --input samplesheet.csv \
    --protocol DNA \
    --input_path ./fast5/ \
    --flowcell FLO-MIN106 \
    --kit SQK-LSK109 \
    --barcode_kit SQK-PBK004 \
    -profile docker

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.

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/nanoseq

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/nanoseq releases page and find the latest version number - numeric only (eg. 1.3.1). Then specify this when running the pipeline with -r (one hyphen) - eg. -r 1.3.1.

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.

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, Podman, 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.

• docker
  • A generic configuration profile to be used with Docker
  • Pulls software from Docker Hub: nfcore/nanoseq
• singularity
  • A generic configuration profile to be used with Singularity
  • Pulls software from Docker Hub: nfcore/nanoseq
• podman
  • A generic configuration profile to be used with Podman
  • Pulls software from Docker Hub: nfcore/nanoseq
• test
  • A profile with a complete configuration for automated testing
  • Includes links to test data so needs no other parameters

-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: star {
    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 above). 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.

Running in the background

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

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.

Alternatively, you can use screen / tmux or similar tool to create a detached session which you can log back into at a later time. Some HPC setups also allow you to run nextflow within a cluster job submitted your job scheduler (from where it submits more jobs).

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'