git clone https://github.com/thibaultdestanque/RNA_seq_Nextflow.git
Import univec.fasta and put it in Other_files
directory.
wget Other_files/univec.fasta ftp://ftp.ncbi.nlm.nih.gov/pub/UniVec/UniVec
Import your genome and its annotation (gff3) and copy it in Ref_Genome
directory
cp Path/to/your/genome/genome.fa Directory/where/you/clone/github/directory/Ref_Genome/genome.fa
cp Path/to/your/annotation/annotation.gff3 Directory/where/you/clone/github/directory/Ref_Genome/annotation.gff3
Import your fastq files to analyse and put them in Fastq
Directory
cp Path/to/your/fastq/files/*fastq Directory/where/you/clone/github/directory/Fastq/*fastq
To launch the pipeline, simply go to the directory My_folder
(see below) and type:
nextflow run main.nf
main.nf
is the nextflow pipeline with all tools and commands launched. Parameters are stored and can be changed in nextflow.config
.
Tree folders must look like this:
My_folder
main.nf
nextflow.config
Ref_Genome
genome.fa
genomeIndex
- 'genome index files'
genome_annotation.gff3
Fastq
fastq files to analyse
Other_files
univec.fa
-Folders/files
that must be present before launching the script
Trimming reads tool.
http://www.usadellab.org/cms/index.php?page=trimmomatic Bolger, Anthony M., Marc Lohse, and Bjoern Usadel. 2014. “Trimmomatic: A Flexible Trimmer for Illumina Sequence Data.” Bioinformatics 30 (15): 2114–20. https://doi.org/10.1093/bioinformatics/btu170.
Indexing and alignment tool.
Wu, Thomas D., Jens Reeder, Michael Lawrence, Gabe Becker, and Matthew J. Brauer. 2016. “GMAP and GSNAP for Genomic Sequence Alignment: Enhancements to Speed, Accuracy, and Functionality.” In Statistical Genomics: Methods and Protocols, edited by Ewy Mathé and Sean Davis, 283–334. Methods in Molecular Biology. New York, NY: Springer New York. https://doi.org/10.1007/978-1-4939-3578-9_15.
Count of reads per genes.
Falini, Giuseppe, and Simona Fermani. 2004. “Chitin Mineralization.” Tissue Engineering 10 (1–2): 1–6. https://doi.org/10.1089/107632704322791646.
Version 0.36
Used in Trim process of main.nf: conda 'bioconda::trimmomatic=0.36'
.
Version 2018.07.04
Used in Index_Genome and Alignment process of main.nf: conda 'bioconda::gmap=2018.07.04'
Version 0.6.1
Used in Htseq_count process of main.nf: conda 'bioconda::htseq=0.6.1'
Using RNAseqAnalysis_last_annotation.R script