RNA sequencing (RNA-Seq) is a powerful technique useful to understand different biological questions, from gene expression and regulation to population genetics and evolution. Usually, the standard protocol begins with the extraction of RNA and its conversion to cDNA in order to be sequenced using high-throughput technologies. The most often used application of RNA-Seq is analyzing differential gene expression (DGE), i.e., comparing the expression of all the genes transcribed under two different conditions. DGE analysis involved three mainly three steps:

1. Map the reads for each sample to the reference genome.
2. Quantify expressed genes and transcripts.
3. Statistical analysis of gene expression.

The Sequence Read Archive (SRA, https://www.ncbi.nlm.nih.gov/sra/) is a public database that stores high-throughput sequencing data to hence the open science phylosophy.

Toy example: Compare the gene expression between human males and females

Step 1: Download the reads to analyse the gene expression. The reads can be downloaded from SRA using download_reads.R. The reads corresponding to chrX from these samples can be found at: /mnt/netapp1/share_miba/rnaseq/reads and the samples information is stored in the following file /mnt/netapp1/share_miba/rnaseq/samples_info.tsv.

Usage: uvigo_transcriptome/download_reads.R [options]

Options:
    -a ACC, --acc=ACC
        Accession number
    -o OUT, --out=OUT
        Output directory
    -c CPU, --cpu=CPU
        Number of threads to use
    -h, --help
        Show this help message and exit

for RUN in $(awk '{print $1}' samples_info.tsv)
do
  Rscript download_reads.R -a $RUN -o /path/reads_dir -c THREADS
done

Run accession	Sex
ERR204889	Male
ERR204966	Male
ERR204995	Male
ERR188273	Female
ERR188060	Female
ERR204896	Female

Step 2: Download and build the index of the human chromosome X (chrX) using get_reference.R. This scripts can be used to fetch any chromosome (–seq chr##) or the whole genome (–seq genome) and build their index.

Usage: uvigo_transcriptome/get_reference.R [options]

Options:
    -s SEQ, --seq=SEQ
        Sequence to download
    -o OUT, --out=OUT
        Output file in FASTA fomat (.fasta)
    -a ANN, --ann=ANN
        Download the annotation of the sequence
    -p CDS, --cds=CDS
        Extract protein coding sequences
    -h, --help
        Show this help message and exit

Rscript get_reference.R -s chrX -o /path/ref_dir -a TRUE -p TRUE

Step 3: Quantify the gene expression and analyse the differences between both groups. Use expression_quant.R to perform both tasks.

Usage: uvigo_transcriptome/expression_quant.R [options]

Options:
    -r REF, --ref=REF
        Reference sequence (.fasta)
    -a ANN, --ann=ANN
        Reference annotation (.gtf)
    -p SPL, --spl=SPL
        Samples information
    -o OUT, --out=OUT
        Output directory
    -t TEC, --tec=TEC
        Type of reference used: genome (geno) or transcriptome (tran)
    -c CPU, --cpu=CPU
        Number of threads to use
    -h, --help
        Show this help message and exit

Rscript expression_quant.R -r /path/ref_seq.fasta -a /path/ref_ann.gtf -p /path/sample_info.tsv -o /path/out_dir -t geno -c THREADS