GSE124439 Processing Pipeline

Publication

Acta neuropathologica (2022) — PMID 35778567

Dataset

Postmortem Cortex Samples Identify Distinct Molecular Subtypes of ALS: Retrotransposon Activation, Oxidative Stress, and Activated Glia

1. 1

   fastq Illumina RNASeq paired-end reads were aligned to the hg19 reference genome using STAR v2.5.2b with the following configurations: --outFilterMultimapNmax 100; --outFilterMismatchNoverReadLmax 0.04

   STAR v2.5.2b GitHub

   $ Bash example

   # Install STAR (if not already installed)
   # conda install -c bioconda star=2.5.2b
   
   # Define input and output files
   READ1="sample_R1.fastq.gz" # Placeholder for forward reads
   READ2="sample_R2.fastq.gz" # Placeholder for reverse reads
   GENOME_DIR="/path/to/hg19_star_index" # Placeholder for indexed hg19 genome
   OUTPUT_DIR="star_alignment_output"
   NUM_THREADS=8 # Example number of threads
   
   # Create output directory if it doesn't exist
   mkdir -p "${OUTPUT_DIR}"
   
   # Run STAR alignment for paired-end RNA-Seq reads
   STAR --genomeDir "${GENOME_DIR}" \
        --readFilesIn "${READ1}" "${READ2}" \
        --runThreadN "${NUM_THREADS}" \
        --outFileNamePrefix "${OUTPUT_DIR}/" \
        --outFilterMultimapNmax 100 \
        --outFilterMismatchNoverReadLmax 0.04 \
        --outSAMtype BAM SortedByCoordinate \
        --outSAMunmapped Within \
        --outSAMattributes Standard \
        --readFilesCommand zcat # Use zcat for gzipped fastq files
   

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2. 2

   Genes and transposable elements (TE) were quantified using TEcount (from TEtranscripts v2.0.3) with the following configurations: hg19 genic GTF from refGene (Jan 2018), custom hg19 TE GTF generated from repeatMasker; --stranded reverse

   TEcount v2.0.3

   $ Bash example

   # Install TEtranscripts (which includes TEcount)
   # conda install -c bioconda tetranscripts
   
   # Placeholder for hg19 genic GTF from refGene (Jan 2018)
   # This file would typically be generated from UCSC Table Browser or a similar resource.
   GENE_GTF="path/to/hg19_refGene_genes.gtf"
   
   # Placeholder for custom hg19 TE GTF generated from RepeatMasker
   # This file would be custom-generated by the user based on RepeatMasker output.
   TE_GTF="path/to/hg19_repeatmasker_TEs.gtf"
   
   # Input BAM file(s) (replace with actual input file paths)
   INPUT_BAM="sample1.bam"
   
   # Output file for quantification results
   OUTPUT_COUNTS="quantification_results.tsv"
   
   # Execute TEcount for quantification of genes and transposable elements
   TEcount --format BAM --mode multi --stranded reverse --GTF "${GENE_GTF}" --TE "${TE_GTF}" -o "${OUTPUT_COUNTS}" "${INPUT_BAM}"

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Tools Used