Introduction Usage Output

Usage

Table of contents

General Nextflow info

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.

It is recommended to limit the Nextflow Java virtual machines memory. We recommend adding the following line to your environment (typically in ~/.bashrc or ~./bash_profile):

NXF_OPTS='-Xms1g -Xmx4g'

Running the pipeline

The typical command for running the pipeline is as follows:

nextflow run nf-core/lncpipe --reads '*_R{1,2}.fastq.gz' -profile standard,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.

Prepare Input file

  • Reference files for humans

    1. hisat index built from Genome: http://cancerbio.info/pub/hg38_hisat_index.tar.gz

    2. Genome reference: ftp://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_human/release_27/GRCh38.p10.genome.fa.gz

    3. GENCODE gene annotation: ftp://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_human/release_27/gencode.v27.annotation.gtf.gz

    4. LNCipedia gene annotation: https://lncipedia.org/downloads/lncipedia_5_0_hc_hg38.gtf

    5. Raw sequence file with *.fastq.gz / *.fq.gz suffixed

    6. design file (optional)

  • Reference files for mouse

    1. hisat index built from Genome

    2. Genome reference:
      ftp://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_mouse/release_M16/GRCm38.p5.genome.fa.gz

    3. GENCODE gene annotation: ftp://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_mouse/release_M16/gencode.vM16.annotation.gtf.gz

    4. Raw sequence file with *.fastq.gz / *.fq.gz suffixed

    5. design file (optional)

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/lncpipe

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/lncpipe 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.

Main Arguments

-profile

Use this parameter to choose a configuration profile. Profiles can give configuration presets for different compute environments. Note that multiple profiles can be loaded, for example: -profile standard,docker - the order of arguments is important!

  • standard
    • The default profile, used if -profile is not specified at all.
    • Runs locally and expects all software to be installed and available on the PATH.
  • docker
    • A generic configuration profile to be used with Docker
    • Pulls software from dockerhub: nfcore/lncpipe
  • singularity
    • A generic configuration profile to be used with Singularity
    • Pulls software from singularity-hub
  • conda
    • A generic configuration profile to be used with conda
    • Pulls most software from Bioconda
  • awsbatch
    • A generic configuration profile to be used with AWS Batch.
  • test
    • A profile with a complete configuration for automated testing
    • Includes links to test data so needs no other parameters
    • NOTE: in lncPipe, test data should be downloaded separately, plz see details in here
  • none
    • No configuration at all. Useful if you want to build your own config from scratch and want to avoid loading in the default base config profile (not recommended).

--reads

Use this to specify the location of your input FastQ files. For example:

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

Please note the following requirements:

  1. The path must be enclosed in quotes
  2. The path must have at least one * wildcard character
  3. When using the pipeline with paired end data, the path must use {1,2} notation to specify read pairs.

If left unspecified, a default pattern is used: data/*{1,2}.fastq.gz

--singleEnd

By default, the pipeline expects paired-end data. If you have single-end data, you need to specify --singleEnd on the command line when you launch the pipeline. A normal glob pattern, enclosed in quotation marks, can then be used for --reads. For example:

--singleEnd --reads '*.fastq'

It is not possible to run a mixture of single-end and paired-end files in one run.

Reference Genomes

The lncPipe currently support human species only, other species will be supported in the near feature.

We recommended users adopted the reference file from GENCODE

Annotation file

--gencode_annotation_gtf

An annotation file from GENCODE database for annotating lncRNAs(required if not set in config file). e.g. gencode.v26.annotation.gtf

--lncipedia_gtf

An annotation file from LNCipedia database for annotating lncRNAs(required if not set in config file) e.g. lncipedia_4_0_hc_hg38.gtf |

--star_index/--bowtie2_index/--hisat2_index

This parameter is required when not configured in nextflow.config file. It specify the star/tophat/hisat2(mutually exclusive) index folder built before running LncPipe . If you don’t know what it is?You can use --fasta to specify the reference sequence data. The index file would be built by LncPipe automatically.

--fasta

If you prefer, you can specify the full path to your reference genome when you run the pipeline:

--fasta '[path to Fasta reference]'

--design

Experimental design file matrix for differential expression analysis. Default: null Format:

WT:Sample1,Sample2,Sample3
KO:Sample1,Sample2,Sample3

While KO/WT represents the two experimental condition, and sample1, sample2, sample3 are replicates which should be comma-delimited in the same line .

For sample names, it should be the sample as the prefix of fastq files which was trimmed by --fastq_ext.

For example:

if fastq file names are Sample1_1.fq.gz, Sample1_2.fq.gz that comes from one sample and your --fastq_ext is set as *_{1,2}.fq.gz, the sample name should be Sample1.

Job Resources

Automatic resubmission

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.

Custom resource requests

Wherever process-specific requirements are set in the pipeline, the default value can be changed by creating a custom config file. See the files in conf for examples.

AWS Batch specific parameters

Running the pipeline on AWS Batch requires a couple of specific parameters to be set according to your AWS Batch configuration. Please use the -awsbatch profile and then specify all of the following parameters.

--awsqueue

The JobQueue that you intend to use on AWS Batch.

--awsregion

The AWS region to run your job in. Default is set to eu-west-1 but can be adjusted to your needs.

Please make sure to also set the -w/--work-dir and --outdir parameters to a S3 storage bucket of your choice - you’ll get an error message notifying you if you didn’t.

Other command line parameters

--outdir

The output directory where the results will be saved.

--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 speicfy this on the command line for every run.

-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)

-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.

NB: Single hyphen (core Nextflow option)

-c

Specify the path to a specific config file (this is a core NextFlow command).

NB: Single hyphen (core Nextflow option)

Note - you can use this to override defaults. For example, you can specify a config file using -c that contains the following:

process.$multiqc.module = []

--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’“

--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'

--max_cpus

Use to set a top-limit for the default CPU requirement for each process. Should be a string in the format integer-unit. eg. --max_cpus 1

--plaintext_email

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

### --multiqc_config Specify a path to a custom MultiQC configuration file.
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