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README.md

RNAja

PythonVersions SnakemakeVersions Singularity

About RNAja

RNAja is a pipeline written in snakemake, allowing to analyse RNAseq data, perform differential expression analysis using several mappers and counters. RNAja is the only pipeline able to compare method effects over DE genes.

1. Install dependencies and clone RNAja

Check dependencies for RNAja : Python < 3.12.0 and Snakemake >= 7.32.4

Here, you can install RNAja from the gitlab source code such as :

    git clone git@forge.ird.fr:phim/rnaja-pipeline.git
    cd rnaja-pipeline
    python3 -m pip install .

Don't forget to export RNAja into the $PATH environment path by

    export PATH=/home/$USER/.local/bin/:$PATH`

1.1 Installing in local mode

   RNAja install_local --help
   RNAja install_local

1.2 Installing in cluster mode

Install RNAja in cluster mode using singularity container from rnaja_utilities https://itrop.ird.fr/RNAja_utilities/

   RNAja install_cluster --help
   RNAja install_cluster --scheduler slurm --env singularity

Please run command line 'RNAja edit_cluster_config' before the first run of RNAja see https://forge.ird.fr/phim/rnaja-pipeline/README.md and configure by default threads and memory resources.

2. Running a datatest

Running test with a datatest from RNAja_utilities in a repertory TEST

   RNAja test_install --help
   RNAja test_install -d TEST

2.1 Running your data in LOCAL mode

launching suggested command line done by RNAja, in LOCAL mode:

   RNAja run_local --help
   RNAja run_local -t 8 -c TEST/data_test_config.yaml --singularity-args "--bind $HOME"

In local mode, it's possible to allocate threads to some rules using --set-threads snakemake argument such as

    RNAja run_local -t 8 -c TEST/data_test_config.yaml --set-threads hisat2_index=4 hisat2_map_paired=2

2.2 Running your data in CLUSTER mode

Now you can launch suggested command line done by RNAja in cluster mode BUT before you need to configurate cluster parameters running command line RNAja edit_cluster_config before the first run and modify threads, ram, node and computer resources. RNAja does a copy of cluster_config.yaml file into your home "/home/$USER/.config/rnaja_pipeline/cluster_config.yaml"

   RNAja run_cluster --help 
   RNAja edit_cluster_config

If singularity was selected in installation of RNAja, it could be needed to give argument --singularity-args \"--bind $HOME\" to Snakemake, by using :

   RNAja run_cluster --help
   RNAja run_cluster -c TEST/data_test_config.yaml --singularity-args "--bind $HOME"
   
   # here a example adapted to @IFB HPC
   RNAja run_cluster -c TEST/data_test_config.yaml --singularity-args "--bind /shared:/shared"
   
   #you can also use snakemake parameters as 
   RNAja run_cluster -c TEST/data_test_config.yaml --singularity-args "--bind $HOME" --rerun-incomplete --nolock

Important Note : In IRD "i-Trop" cluster, run RNAja using ONLY a node, data has to be in "/scratch" of chosen node. Use nodelist : nodeX parameter inside of cluster_config.yaml̀ file.

3. Running your data

3.1.create a pipeline by configuring the config.yaml file

Before to run RNAja, create a config.yaml by using before to adapt it.

RNAja create_config

Three sections are needed for RNAja into the config.yaml file: section DATA, MODE and PARAMS.

DATA section

Adapt config.yaml file with path to fastq files, reference and annotation file in the DATA section as well as the output directory.

DATA:
    fastq_dir: "/path/to/FASTQ"
    reference: "/path/to/reference/ref.fna"
    annotation: "/path/to/reference/ref.gtf"
    output_dir: "RNAJA_OUTPUT"
    sample_info: "/path/to/sample_info.txt"
    PAIRED : true
    de_comparisons: "/path/to/treatmentsComparisons.csv"

sample_info key

Into the sample_info DATA key, you need to give a comma separated sample_info.txt file containing information about samples. This file needs header with SampleName,Forward,Reverse,Treatment,Experiment columns.

If you are in single mode, don't fill on 'reverse' column in sample_info.txt file

Here an example for experiment in single mode :

SampleName,Forward,Reverse,Treatment,Experiment
Batch-1,/path/to/FASTQ/Batch-rep1_R1.fastq.gz,,Batch,E1
Batch-2,/path/to/FASTQ/Batch-rep2_R1.fastq.gz,,Batch,E2
Batch-3,/path/to/FASTQ/Batch-rep3_R1.fastq.gz,,Batch,E3
CENPK-1,/path/to/FASTQ/CENPK-rep1_R1.fastq.gz,,CENPK,E1
CENPK-2,/path/to/FASTQ/CENPK-rep2_R1.fastq.gz,,CENPK,E2
CENPK-3,/path/to/FASTQ/CENPK-rep3_R1.fastq.gz,,CENPK,E3

Or paired mode ...

SampleName,Forward,Reverse,Treatment,Experiment
Batch-1,/path/to/FASTQ/Batch-rep1_R1.fastq.gz,/path/to/FASTQ/Batch-rep1_R2.fastq.gz,Batch,E1
Batch-2,/path/to/FASTQ/Batch-rep2_R1.fastq.gz,/path/to/FASTQ/Batch-rep2_R2.fastq.gz,Batch,E2
Batch-3,/path/to/FASTQ/Batch-rep3_R1.fastq.gz,/path/to/FASTQ/Batch-rep3_R2.fastq.gz,Batch,E3
CENPK-1,/path/to/FASTQ/CENPK-rep1_R1.fastq.gz,/path/to/FASTQ/CENPK-rep1_R2.fastq.gz,CENPK,E1
CENPK-2,/path/to/FASTQ/CENPK-rep2_R1.fastq.gz,/path/to/FASTQ/CENPK-rep2_R2.fastq.gz,CENPK,E2
CENPK-3,/path/to/FASTQ/CENPK-rep3_R1.fastq.gz,/path/to/FASTQ/CENPK-rep3_R2.fastq.gz,CENPK,E3

Finally, you need confirm if reads are paired or single filling in PAIRED param using true or false boolean. If PAIRED : true, samples suffix should be _R1.fastq.gz and _R2.fastq.gz.

ATTENTION : If yours reads are ilumina paired, you need rename reads SAMPLE_R1.fastq.gz and SAMPLE_R2.fastq.gz. For single reads use SAMPLE_R1.fastq.gz. RNAja uses compressed and decompressed fastq files.

de_comparisons key

In the de_comparisons indicate the path to the treatementsComparaison file. This file is used to differential expression analysis. Please declare treatments you want to compare. Here an example. RNAja expects a header with condA and CondB comma separated columns :

condA,condB
Batch,CENPK

MODE section

Five pipelines can be running in parallel by using RNAja !

We have included for instance two mappers (STAR and HISAT2) and 3 counters (STRINGTIE, HTSEQCOUNT,STAR). You can activate or deactivate pipelines you would run in the MODE section such as ...

MODE:
    HISAT2_STRINGTIE: true
    HISAT2_HTSEQCOUNT: true
    STARmap_STARcount: true
    STARmap_HTSEQCOUNT: true
    STARmap_STRINGTIE: true

PARAMS section

In the PARAMS section, tools parameters can be modified and adapted.

PARAMS:
    HISAT2:
        indexation:
            prefix: "REF"
    STAR:
        indexation:
            params: "--sjdbOverhang 100 --genomeSAindexNbases 10"
        mapping:
            params: "--readFilesCommand zcat" # --outFilterMismatchNoverLmax 0.03
    STRINGTIE:
        discovery_mode : false
    HTSEQCOUNT:
        params: "-r pos -s reverse -m union -t gene "

You can modify HISAT2 indexation, STAR indexation and mapping options, change STRINGTIE mode (discovery or not) as well as HTSEQCOUNT params. Feel free to check documentation of these tools before to run RNAja!

output

Here an example of output_dir if you have activated all five pipelines proposed by RNAja.

RNAJA_OUTPUT/
├── COUNT
│   ├── HTSEQCOUNT
│   │   ├── HISAT2
│   │   └── STAR
│   ├── STAR
│   │   ├── Batch-rep1Aligned.out.bam
│   │   ├── Batch-rep1Aligned.toTranscriptome.out.bam
│   │   ├── Batch-rep1Log.final.out
│   │   ├── Batch-rep1Log.out
│   │   ├── Batch-rep1Log.progress.out
│   │   ├── Batch-rep1ReadsPerGene.out.tab
│   │   ├── Batch-rep1SJ.out.tab
...
│   │   ├── CENPK-rep1Aligned.out.bam
│   │   ├── CENPK-rep1Aligned.toTranscriptome.out.bam
│   │   ├── CENPK-rep1Log.final.out
│   │   ├── CENPK-rep1Log.out
│   │   ├── CENPK-rep1Log.progress.out
│   │   ├── CENPK-rep1ReadsPerGene.out.tab
│   │   ├── CENPK-rep1SJ.out.tab
...
│   └── STRINGTIE
│       ├── HISAT2_Batch-rep1.gtf
│       ├── HISAT2_Batch-rep1.tsv
...
│       ├── HISAT2_CENPK-rep3.gtf
│       ├── HISAT2_CENPK-rep3.tsv
│       ├── STAR_Batch-rep1.gtf
│       ├── STAR_Batch-rep1.tsv
...
│       ├── STAR_CENPK-rep3.gtf
│       ├── STAR_CENPK-rep3.tsv
├── LOGS
├── MAPPING
│   ├── HISAT2
│   │   ├── Batch-rep1.bam
│   │   ├── Batch-rep1.bam.csi
│   │   ├── Batch-rep1_HISAT_summary.txt
...
│   │   ├── CENPK-rep3.bam
│   │   ├── CENPK-rep3.bam.csi
│   │   └── CENPK-rep3_HISAT_summary.txt
│   └── STAR
│       ├── Batch-rep1.bam
│       ├── Batch-rep1.bam.csi
...
│       ├── CENPK-rep3.bam
│       └── CENPK-rep3.bam.csi
├── REF
│   ├── HISAT2
│   │   ├── GCF_000146045.2_R64_genomic.fasta -> /scratch/rnaja_test/TEST/DATA_TEST/REF/GCF_000146045.2_R64_genomic.fna
│   └── STAR
│       ├── chrLength.txt
│       ├── chrNameLength.txt
│       ├── chrName.txt
│       ├── chrStart.txt
│       ├── exonGeTrInfo.tab
│       ├── exonInfo.tab
│       ├── GCF_000146045.2_R64_genomic.fasta -> /scratch/rnaja_test/TEST/DATA_TEST/REF/GCF_000146045.2_R64_genomic.fna
│       ├── geneInfo.tab
│       ├── Genome
│       ├── genomeParameters.txt
│       ├── Log.out
│       ├── SA
│       ├── SAindex
│       ├── sjdbInfo.txt
│       ├── sjdbList.fromGTF.out.tab
│       ├── sjdbList.out.tab
│       └── transcriptInfo.tab
└── slurm_logs

Authors

  • Aurore Comte (IRD), Christine Tranchant (IRD), Julie Orjuela (IRD)

Some parts of RNAja code and documentation were inspired or came from the pipelines below.

RNAja uses really nice python package SnakEcdysis https://snakecdysis.readthedocs.io/en/latest/package.html to perform installation and execution in local and cluster mode. SnakEcdysis is developed by Sébastien Ravel (CIRAD).

Acknowledgements

Thanks to Ndomassi Tando (i-Trop IRD) by administration support.

The authors acknowledge the IRD i-Trop HPC https://bioinfo.ird.fr/ (South Green Platform http://www.southgreen.fr) at IRD Montpellier for providing HPC resources that have contributed to this work.

Thanks to Alexis Dereeper for his help and the development of diffexDB https://bioinfo-web.mpl.ird.fr/cgi-bin2/microarray/public/diffexdb.cgi.

License

Licenced under MIT https://opensource.org/license/mit/. Intellectual property belongs to IRD and authors.