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A RNAseq pipeline from raw reads to feature counts

Project description

This is is the rnaseq pipeline from the Sequana projet


RNASeq analysis from raw data to feature counts


A set of Fastq Files and genome reference and annotation.


MultiQC reports and feature Counts




Cokelaer et al, (2017), ‘Sequana’: a Set of Snakemake NGS pipelines, Journal of Open Source Software, 2(16), 352, JOSS DOI doi:10.21105/joss.00352



You must install Sequana first:

pip install sequana

Then, just install this package:

pip install sequana_rnaseq


sequana_pipelines_rnaseq --help
sequana_pipelines_rnaseq --input-directory DATAPATH --genome-directory genome --aligner star

This creates a directory with the pipeline and configuration file. You will then need to execute the pipeline:

cd rnaseq
sh  # for a local run

This launch a snakemake pipeline. If you are familiar with snakemake, you can retrieve the pipeline itself and its configuration files and then execute the pipeline yourself with specific parameters:

snakemake -s rnaseq.rules -c config.yaml --cores 4 --stats stats.txt

Or use sequanix interface.


This pipelines requires the following executable(s):

  • bowtie
  • bowtie2
  • STAR
  • featureCounts (subread package)
  • picard
  • multiqc

More may be needed depending on the configuration file options. For instance, you may use fastq_screen, in which case you need to install it and configure it.


This pipeline runs rnaseq in parallel on the input fastq files (paired or not). A brief sequana summary report is also produced.

Rules and configuration details

Here is the latest documented configuration file to be used with the pipeline. Each rule used in the pipeline may have a section in the configuration file.


the RNAseQC rule is switch off and is not currently functional in version 0.9.X


Version Description
  • Remove the try/except around tolerance (guess of strandness) to make sure this is provided by the user. Final onsuccess benefits from faster GFF function (sequana 0.9.4)
  • Fix typo (regression bug) + add tolerance in schema + generic title in multiqc_config. (oct 2020)
  • add the tolerance parameter in the feature_counts rule as a user parameter (config and pipeline).
  • Best feature_counts is now saved into rnadiff/feature_counts directory and rnadiff scripts have been updated accordingly
  • the most probable feature count option is now computed more effectivily and incorporated inside the Snakemake pipeline (not in the onsuccess) so that multiqc picks the best one (not the 3 results)
  • the target.txt file can be generated inside the pipeline if user fill the rnadiff/conditions section in the config file
  • indexing options are filled automatically when calling sequana_rnaseq based on the presence/absence of the index of the aligner being used.
  • salmon now integrated and feature counts created (still WIP in sequana)
  • FastQC on raw data skipped by default (FastQC for processed data is still available)
  • Added paired options (-p) for featureCounts
  • Switch back markduplicates to False for now.
  • Use only R1 with bowtie1
  • set the memory requirements for mark_duplicates in cluster_config file
  • Set temporary directory for mark_duplicates to be local ./tmp
  • set mark_duplicate to true by default
  • use new sequana pipeline manager
  • export all features counts in a single file
  • custom HTML report
  • faster –help calls
  • –from-project option added
  • include salmon tool as an alternative to star/bowtie2
  • include rnadiff directory with required input for Differential analysis
  • Automatic guessing of the strandness of the experiment
  • Fix multiqc for RNAseQC rule
  • Fix RNAseQC rule, which is now available.
  • Fix ability to use existing rRNA file as input
  • Fix indexing for bowtie1 to not be done if aligner is different
  • add new options: –feature-counts-options and –do-rnaseq-qc, –rRNA-feature
  • Based on the input GFF, we now check the validity of the rRNA feature and feature counts options to check whether the feature exists in the GFF
  • schema is now used to check the config file values
  • add a data test for testing and documentation
  • fix typo found in version 0.9.6
  • Fixed empty read tag in the configuration file
  • Possiblity to switch off cutadapt section
  • Fixing bowtie2 rule in sequana and update the pipeline accordingly
  • Include a schema file
  • output-directory parameter renamed into output_directory (multiqc section)
  • handle stdout correctly in fastqc, bowtie1, bowtie2 rules
0.9.3 if a fastq_screen.conf is provided, we switch the fastqc_screen section ON automatically

Major refactorisation.

  • remove sartools, kraken rules.
  • Indexing is now optional and can be set in the configuration.
  • Configuration file is simplified with a general section to enter the genome location and aligner.
  • Fixed rules in sequana (0.8.0) that were not up-to-date with several executables used in the pipeline including picard, fastq_screen, etc. See Sequana Changelog for details with respect to rules changes.
  • Copying the feature counts in main directory ready to use for a differential analysis.

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