/RNA_seq_Nextflow

Pipeline for RNA seq analysis

Primary LanguageR

RNAseq pipeline analysis

Clone git hub directory

git clone https://github.com/thibaultdestanque/RNA_seq_Nextflow.git

Import univec

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 Genome and annotation

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 Fastq

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

Launch pipeline

To launch the pipeline, simply go to the directory My_folder (see below) and type:

nextflow run main.nf

Documentation

Scripts

main.nf is the nextflow pipeline with all tools and commands launched. Parameters are stored and can be changed in nextflow.config.

Working directory

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

Steps:

Trimmomatic

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.

Gsnap

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.

Htseq count

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.

Conda

Trimmomatic

Version 0.36

Used in Trim process of main.nf: conda 'bioconda::trimmomatic=0.36'.

Gsnap

Version 2018.07.04

Used in Index_Genome and Alignment process of main.nf: conda 'bioconda::gmap=2018.07.04'

Htseq-count

Version 0.6.1

Used in Htseq_count process of main.nf: conda 'bioconda::htseq=0.6.1'

RNA seq analysis with R

Using RNAseqAnalysis_last_annotation.R script