Tuxedo-NF

A Nextflow implementation of the Tuxedo Suite of Tools Workflow is based on the 2016 Nature Protocols publication: "Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown"

Quick start

Make sure you have all the required dependencies listed in the last section.

Install the Nextflow runtime by running the following command:

$ curl -fsSL get.nextflow.io | bash

When done, you can launch the pipeline execution by entering the command shown below:

$ nextflow run skptic/tuxedo-nf

By default the pipeline is executed against the provided example dataset. Check the Pipeline parameters section below to see how enter your data on the program command line.

All parameters can be specified at the command line or alternatively specified in a parameters config file.

Default parameters can be found in the params_default.config file.

Pipeline parameters

--reads

• Specifies the local location of the reads fastq file(s).
• Multiple files can be specified using the usual wildcards (*, ?), in this case make sure to surround the parameter string value by single quote characters (see the example below)
• It must end in .fastq or fastq.gz.
• See --sra_ids and --use_sra to pull reads directly from the NCBI SRA.
• Involved in the task mapping.
• By default it is set to the Tuxedo-NF's location: ./example-data/reads/SRR*_*_{1,2}.fastq.gz

Example:

$ nextflow run skptic/tuxedo-nf --reads '/home/dataset/*.fastq'

This will handle each fastq file as a seperate sample.

Read pairs of samples can be specified using the glob file pattern. Consider a more complex situation where there are three samples (A, B and C), with A and B being paired reads and C being single ended. The read files could be:

sample_A_1.fastq
sample_A_2.fastq
sample_B_1.fastq
sample_B_2.fastq 
sample_C_1.fastq

The reads may be specified as below:

$ nextflow run skptic/tuxedo-nf --reads '/home/dataset/sample_*_{1,2}.fastq'    

--genome

• The location of the genome multi-fasta file.
• It should end in .fa
• See --genome_address and --download_genome to pull a genome directly.
• Involved in the task: genome_index.
• By default it is set to the Tuxedo-NF's localization: ./example-data/genome/genome.fa

Example:

$ nextflow run skptic/tuxedo-nf --genome /home/user/my_genome/example.fa

--index

• The location of a HISAT2 Index.
• It should point to the location of the index (without the .X.ht2 suffix)
• See --run_index to have the index generated from a genome.
• Involved in the task: genome_index.
• By default it is set to the Tuxedo-NF's localization: ./example-data/index/genome

Example:

$ nextflow run skptic/tuxedo-nf --genome /home/user/my_genome_index/example

--pheno

• The location of the phenotype/group description file.

• It should end in .csv or .txt.

• This file defines the groups that each sample belong to.

• ids should be the sample name, for example the SRA id or the name of the fastq (without _1.fastq the suffix)

• Usually a tab or comma delimited file, for example:

  ids groups SRR control SRR control SRR disease SRR disease

• Involved in the task: ballgown.

• By default it is set to the Tuxedo-NF's localization: ./example-data/pheno/exp-info.txt

Example:

$ nextflow run skptic/tuxedo-nf --transcriptome /home/user/my_exp/exp-info.txt

--download_genome

• Boolean value [ true || false ]
• If true, the file specified by --genome_address will be downloaded and used for subsequent tasks
• Involved in the tasks: download_genome
• By default --download_genome=false

Example:

$ nextflow run skptic/tuxedo-nf --download-genome=true 

or equivalently just $ nextflow run skptic/tuxedo-nf --download-genome

--genome_address

• http or ftp address specifying a genome in fasta format.
• It should end in .gz or .tar.gz
• Sources for genomes include for ensembl and UCSC.
• If --download_genome is true then the genome will be downloaded from the above address and used for subsequent tasks
• Involved in the tasks: download_genome
• By default --genome_address=ftp://ftp.ensembl.org/pub/release-86/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna_sm.primary_assembly.fa.gz

Example:

$ nextflow run skptic/tuxedo-nf --genome_address=ftp://hgdownload.cse.ucsc.edu/goldenPath/currentGenomes/Homo_sapiens/bigZips/chromFa.tar.gz

--download_annotation

• Boolean value [ true || false ]
• If true, the file specified by --annotation_address will be downloaded and used for subsequent tasks
• Involved in the tasks: download_annotation
• By default --download_annotation=false

Example:

$ nextflow run skptic/tuxedo-nf --download_annotation=true

or equivalently just $ nextflow run skptic/tuxedo-nf --download_annotation

--annotation_address

• http or ftp address specifying an annotation gtf format
• It should end in .gz or tar.gz.
• Sources for genomes include for ensembl and UCSC.
• If --download-annotation is true then the annotation will be downloaded from the above address and used for subsequent tasks
• Involved in the tasks: download_annotation
• By default --annotation_address=ftp://ftp.ensembl.org/pub/release-86/gtf/homo_sapiens/Homo_sapiens.GRCh38.86.gtf.gz

Example:

$ nextflow run skptic/tuxedo-nf --genome_address=ftp://hgdownload.cse.ucsc.edu/goldenPath/currentGenomes/Homo_sapiens/bigZips/chromFa.tar.gz

--run_index

• Boolean value [ true || false ]
• If true, and the file specified by --genome or --genome_address will be indexed and used for subsequent tasks
• If --download_genome is true, then --run_index must be true
• Involved in the tasks: genome_index
• By default --run_index=true

Example:

$ nextflow run skptic/tuxedo-nf --run_index=false

--use_sra

• Boolean value [ true || false ]
• If true, the SRA IDs specified by --sra_ids will be prefectched and used for subsequent tasks
• Involved in the tasks: sra_prefetch
• By default --use_sra=false

Example:

$ nextflow run skptic/tuxedo-nf --use_sra=true

or equivalently just $ nextflow run skptic/tuxedo-nf --use_sra

--sra_ids

• Comma seperated list of SRA reads using Run accession id (ussually SRR or HRR)
• See the NCBI Sequence Read Archive to more information or to browse data.
• If --use-sra is true then the SRA ids listed will be prefetched from either the local cache (see --cache) or downloaded.
• Involved in the tasks: sra_prefetch
• By default --sra_ids="ERR188044,ERR188104,ERR188234,ERR188245,ERR188257,ERR188273,ERR188337,ERR188383,ERR188401,ERR188428,ERR188454,ERR204916"

Example:

$ nextflow run skptic/tuxedo-nf --sra_ids=`SRR349706,SRR349707,SRR349708`

--cache

• Location of NCBI cache.
• If --use-sra is true then the SRA ids listed will be prefetched and stored in the --cache location.
• It should contain a directory called ncbi which contains the sra-tools/vdb database.
• Can be useful if several pipelines use the same input sequences, saving on storage and bandwidth.
• Involved in the tasks: sra_prefetch
• By default --cache=./cache

Example:

$ nextflow run skptic/tuxedo-nf --cache=`/your/ncbi_cache_location`

--output

• Specifies the folder where the results will be stored for the user.
• It does not matter if the folder does not exist.
• By default is set to Tuxedo-NF's folder: ./results

Example:

$ nextflow run skptic/tuxedo-nf --output /home/user/my_results 

Cluster support

Tuxedo-NF execution relies on Nextflow framework which provides an abstraction between the pipeline functional logic and the underlying processing system.

Thus it is possible to execute it on your computer or any cluster resource manager without modifying it.

Currently the following platforms are supported:

• Oracle/Univa/Open Grid Engine (SGE)
• Platform LSF
• SLURM
• PBS/Torque

By default the pipeline is parallelized by spanning multiple threads in the machine where the script is launched.

To submit the execution to a SGE cluster create a file named nextflow.config, in the directory where the pipeline is going to be launched, with the following content:

process {
  executor='sge'
  queue='<your queue name>'
}

In doing that, tasks will be executed through the qsub SGE command, and so your pipeline will behave like any other SGE job script, with the benefit that Nextflow will automatically and transparently manage the tasks synchronisation, file(s) staging/un-staging, etc.

Alternatively the same declaration can be defined in the file $HOME/.nextflow/config.

To lean more about the avaible settings and the configuration file read the Nextflow documentation.

Dependencies

• Java 7+
• Nextflow (0.22.0 or higher)
• Docker