GSE104500 Processing Pipeline

Publication

Nature structural & molecular biology (2017) — PMID 29106412

Dataset

RNA-Seq of L4 C. elegans

1. 1

   Read counts were quantified using kallisto.

   kallisto v0.46.0 GitHub

   $ Bash example

   # Install kallisto (if not already installed)
   # conda install -c bioconda kallisto=0.46.0
   
   # Define variables (replace with actual paths and filenames)
   TRANSCRIPTOME_FASTA="GRCh38_transcriptome.fasta" # Placeholder: Replace with path to your transcriptome FASTA (e.g., from Ensembl or GENCODE)
   KALLISTO_INDEX="kallisto_index.idx"
   SAMPLE_R1_FASTQ="sample_R1.fastq.gz" # Replace with your R1 FASTQ file
   SAMPLE_R2_FASTQ="sample_R2.fastq.gz" # Replace with your R2 FASTQ file (omit if single-end)
   OUTPUT_DIR="kallisto_quant_output"
   NUM_THREADS=8 # Number of threads to use
   
   # 1. Build kallisto index (if not already built)
   # This step needs to be run once for a given transcriptome.
   # kallisto index -i ${KALLISTO_INDEX} ${TRANSCRIPTOME_FASTA}
   
   # 2. Quantify read counts using kallisto
   # For paired-end reads:
   kallisto quant \
     -i ${KALLISTO_INDEX} \
     -o ${OUTPUT_DIR} \
     --bias \
     --threads ${NUM_THREADS} \
     ${SAMPLE_R1_FASTQ} \
     ${SAMPLE_R2_FASTQ}
   
   # For single-end reads (uncomment and modify if applicable):
   # kallisto quant \
   #   -i ${KALLISTO_INDEX} \
   #   -o ${OUTPUT_DIR} \
   #   --single \
   #   -l 200 \
   #   -s 20 \
   #   --bias \
   #   --threads ${NUM_THREADS} \
   #   ${SAMPLE_R1_FASTQ}
   

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2. 2

   These were then aligned to C. elegans genome WS247.

   STAR (Inferred with models/gemini-2.5-flash) v2.7.10a GitHub

   $ Bash example

   # Install STAR (if not already installed)
   # conda install -c bioconda star
   
   # Define variables
   GENOME_DIR="celegans_WS247_STAR_index"
   GENOME_FASTA="c_elegans.WS247.dna.toplevel.fa.gz" # Placeholder: Download from WormBase FTP for WS247
   GENOME_GTF="c_elegans.WS247.annotations.gtf.gz"   # Placeholder: Download from WormBase FTP for WS247
   READS_R1="input_reads_R1.fastq.gz"               # Placeholder for input forward reads
   READS_R2="input_reads_R2.fastq.gz"               # Placeholder for input reverse reads (if paired-end)
   OUTPUT_PREFIX="aligned_reads"
   
   # --- Reference Data Acquisition (Example - replace with actual download if needed) ---
   # For C. elegans genome WS247, reference files are typically found on WormBase FTP.
   # Example download commands (adjust paths and filenames as necessary):
   # wget ftp://ftp.wormbase.org/pub/wormbase/releases/WS247/species/c_elegans/PRJNA13758/c_elegans.PRJNA13758.WS247.dna.toplevel.fa.gz
   # wget ftp://ftp.wormbase.org/pub/wormbase/releases/WS247/species/c_elegans/PRJNA13758/c_elegans.PRJNA13758.WS247.annotations.gtf.gz
   
   # 1. Create STAR genome index
   mkdir -p ${GENOME_DIR}
   STAR --runMode genomeGenerate \
        --genomeDir ${GENOME_DIR} \
        --genomeFastaFiles ${GENOME_FASTA} \
        --sjdbGTFfile ${GENOME_GTF} \
        --sjdbOverhang 100 \
        --runThreadN 8 # Adjust threads as needed
   
   # 2. Align reads to the C. elegans WS247 genome
   # Assuming paired-end reads. For single-end, remove ${READS_R2}
   STAR --runMode alignReads \
        --genomeDir ${GENOME_DIR} \
        --readFilesIn ${READS_R1} ${READS_R2} \
        --outFileNamePrefix ${OUTPUT_PREFIX}_ \
        --outSAMtype BAM SortedByCoordinate \
        --outSAMunmapped Within \
        --outSAMattributes Standard \
        --runThreadN 8 # Adjust threads as needed
   
   # Rename output file for clarity
   mv ${OUTPUT_PREFIX}_Aligned.sortedByCoord.out.bam ${OUTPUT_PREFIX}.bam

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