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# rnaseq v1.1
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**Rnaseq** workflow , which agree to FAIR principles , was built in Nexflow dsl2 language, with singularity container for used softwares, optimized in terms of computing resources (cpu, memory), and its use on a informatic farm with a slurm scheduler.
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## Install rnaseq flow and build singularity image

Clone rnaseq git and build local singularity image (with system admin rights) based on the provided singularity definition file.

```bash
git clone https://forgemia.inra.fr/lpgp/rnaseq.git
sudo singularity build ./rnaseq/singularity/rnaseq.sif ./rnaseq/singularity/rnaseq.def
```

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## Usage examples

### bulk RNAseq paired end reads using STAR and HTSEQ-count
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```bash
#!/bin/bash
#SBATCH -J rnaseq
#SBATCH --mem 10GB
module load containers/singularity/3.9.9
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module load bioinfo/Nextflow/21.10.6
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nextflow run /work/project/lpgp/Nextflow/rnaseq/ \
-profile slurm \
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--input "${PWD}/raw/*_{1,2}.fastq.gz" \
--genome "${PWD}/genome.fa.gz" \
--gff_gtf "${PWD}/annot.gtf.gz" \
--method "star_htseq-count" \
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--sjdbOverhang 80 \
--clip_r1 12 \
--three_prime_clip_r1 2 \
--clip_r2 12 \
--three_prime_clip_r2 2 \
--out_dir "${PWD}/results"
```

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### bulk RNAseq from single end reads using Salmon in SAF mode
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```bash
#!/bin/bash
#SBATCH -J rnaseq
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#SBATCH --mem 10GB
module load containers/singularity/3.9.9
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module load bioinfo/Nextflow/21.10.6
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nextflow run /work/project/lpgp/Nextflow/rnaseq/ \
-profile slurm \
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--input "${PWD}/raw/*.fastq.gz" \
--genome "${PWD}/genome.fa.gz" \
--transcriptome "${PWD}/transcriptome.fa.gz" \
--method "salmon_saf" \
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--clip_r1 12 \
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--three_prime_clip_r1 2 \
--out_dir "${PWD}/results"
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```

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### bulk RNAseq from paired end reads using Salmon in SAF mode
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```bash
#!/bin/bash
#SBATCH -J rnaseq
#SBATCH --mem 10GB
module load containers/singularity/3.9.9
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module load bioinfo/Nextflow/21.10.6
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nextflow run /work/project/lpgp/Nextflow/rnaseq/ \
-profile slurm \
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--input "${PWD}/raw/*_{1,2}.fastq.gz" \
--method "salmon_saf" \
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--genome "${PWD}/genome.fa.gz" \
--transcriptome "${PWD}/transcriptome.fa.gz" \
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--clip_r1 12 \
--three_prime_clip_r1 2 \
--clip_r2 12 \
--three_prime_clip_r2 2 \
--out_dir "${PWD}/results"
```

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## bulk RNAseq from brb chemistry using STARsolo
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```bash
#!/bin/bash
#SBATCH -J rnaseq
#SBATCH --mem 10GB
module load containers/singularity/3.9.9
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module load bioinfo/Nextflow/21.10.6
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nextflow run /work/project/lpgp/Nextflow/rnaseq/ \
-profile slurm \
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--input "${PWD}/raw/*_{1,2}.fastq.gz" \
--genome "${PWD}/genome.fa.gz" \
--gff_gtf "${PWD}/annot.gtf.gz" \
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--sjdbOverhang 99 \
--whitelist "${PWD}/whitelist.txt" \
--soloCBstart  1 \
--soloCBlen 12 \
--soloUMIstart 13 \
--soloUMIlen 16 \
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--out_dir "${PWD}/results"
```

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## Single cell RNAseq from chromium (v3) chemistry using STARsolo
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```bash
# get The 10X Chromium V3 whitelist
wget https://github.com/10XGenomics/cellranger/raw/master/lib/python/cellranger/barcodes/3M-february-2018.txt.gz

# unzip
gunzip 3M-february-2018.txt.gz
```

```bash
#!/bin/bash
#SBATCH -J rnaseq
#SBATCH --mem 10GB
module load containers/singularity/3.9.9
module load bioinfo/Nextflow/21.10.6
nextflow run /work/project/lpgp/Nextflow/rnaseq/ \
-profile slurm \
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--input "${PWD}/raw/*_{1,2}.fastq.gz" \
--genome "${PWD}/genome.fa.gz" \
--gff_gtf "${PWD}/annot.gtf.gz" \
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--method "star_solo" \
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--whitelist "${PWD}/3M-february-2018.txt" \
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--soloCBstart  1 \
--soloCBlen 16 \
--soloUMIstart 17 \
--soloUMIlen 12 \
--out_dir "${PWD}/results"
```

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## Single cell RNAseq from chromium (v3) chemistry using ALEVIN-fry (through simpleaf)
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```bash
#!/bin/bash
#SBATCH -J rnaseq
#SBATCH --mem 10GB
module load containers/singularity/3.9.9
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module load bioinfo/Nextflow/21.10.6
nextflow run /work/project/lpgp/Nextflow/rnaseq/ \
-profile slurm \
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--input "${PWD}/raw/*_{1,2}.fastq.gz" \
--genome "${PWD}/genome.fa.gz" \
--gff_gtf "${PWD}/annot.gtf.gz" \
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--chemistry "10xv3" \
--unfiltered-pl \
--rlen 91 \
--out_dir "${PWD}/results"
```
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## chemistry options parameters

# chromium 10x v2 chemistry options
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--chemitry "10xv2" # "1{b[16]u[10]x:}2{r:}"
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--rlen 98

# chromium 10x v3 chemistry options
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--chemistry "10xv3" # "1{b[16]u[12]x:}2{r:}"
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--rlen 91

# brb chemistry options (bulk RNAseq)
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--chemistry "brb" # "1{b[12]u[16]x:}2{r:}"
--whitelist "brb_whitelist.txt"
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--rlen 91
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```

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## Defaults parameters
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Please refer to [Trim Galore](https://github.com/FelixKrueger/TrimGalore), [STAR](https://github.com/alexdobin/STAR), [htseq-count](https://htseq.readthedocs.io/en/master/), and
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[Salmon](https://salmon.readthedocs.io/en/latest/salmon.html) [simpleaf](https://simpleaf.readthedocs.io/en/latest/index.html) for complete arguments explanation.
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```bash
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# sequences
input = false
genome = false
transcriptome = false

# fastqc
skip_fastqc = false

# trimming
skip_trimming = false
clip_r1 = 0
clip_r2 = 0
three_prime_clip_r1 = 0
three_prime_clip_r2 = 0

# alignment mode should be star_htseq-count and/or salmon_saf for bulk-RNAseq
# alignment mode should be star_solo and/or alevin_saf and/or alevin_fry for BRBseq or scRNAseq
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method = false

# STAR options
star_index = false
gff_gtf = false
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sjdbOverhang = 99
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keep_star_index = false
htseq_count_multimapped = false
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feature_type = "exon"
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# SALMON options
salmon_index = false
keep_salmon_index = false
writeMappings = false

alevin_fry_index = false
keep_alevin_fry_index = false
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chemistry = false
unfiltered-pl  = false
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# STAR SOLO options
star_index = false
gff_gtf = false
keep_star_index = false
barcodes = false
soloCBstart =  1
soloCBlen = 12
soloUMIstart = 13
soloUMIlen = 16

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# save directory
out_dir = "${PWD}/results"
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```
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## References

1. Krueger F, Galore T. A wrapper tool around cutadapt and FastQC to consistently apply quality and adapter trimming to FastQ files [Internet]. Available from: http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/
2. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29:15–21.
3. Anders S, Pyl PT, Huber W. HTSeq–a Python framework to work with high-throughput sequencing data. Bioinformatics. 2015;31:166–9.
4. Srivastava A, Malik L, Sarkar H, Zakeri M, Almodaresi F, Soneson C, et al. Alignment and mapping methodology influence transcript abundance estimation. Genome Biol. 2020;21:239.
5. Patro R, Duggal G, Love MI, Irizarry RA, Kingsford C. Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods. 2017;14:417–9.