GSE220845 Processing Pipeline

Publication

Molecular cell (2023) — PMID 37084731

Dataset

Proteomic discovery of chemical probes that perturb protein complexes in human cells II

1. 1

   For quantification of alternative RNA splicing, fastq files are adapter-trimmed by cutadapt 3.4 and aligned to GRCh38 by STAR 2.7 .6 and analyzed using rMATS (v4.1.1) (Shen et al., 2014) using the GENCODE (v35) GTF annotation for GRCh38 and the following parameters: -t paired --libType fr-unstranded –readLength 150 --novelSS

   STAR v2.7 GitHub

   $ Bash example

   # Install STAR if not already installed
   # conda install -c bioconda star
   
   # Define variables for input and output
   FASTQ_R1="path/to/your/sample_R1.fastq.gz" # Adapter-trimmed fastq file R1
   FASTQ_R2="path/to/your/sample_R2.fastq.gz" # Adapter-trimmed fastq file R2
   OUTPUT_DIR="path/to/your/output_directory"
   SAMPLE_PREFIX="sample_name" # e.g., SRR1234567
   
   # Reference datasets (GRCh38, GENCODE v35 GTF)
   # Ensure the STAR genome index for GRCh38 (with GENCODE v35) is pre-built.
   # Example command to build index (run once):
   # STAR --runMode genomeGenerate --genomeDir "path/to/your/STAR_genome_index/GRCh38_gencode_v35" \
   #      --genomeFastaFiles "path/to/your/references/GRCh38.primary_assembly.genome.fa" \
   #      --sjdbGTFfile "path/to/your/annotations/gencode.v35.annotation.gtf" \
   #      --sjdbOverhang 149 --runThreadN 16 # Adjust sjdbOverhang to read length - 1 (150-1=149)
   
   GENOME_DIR="path/to/your/STAR_genome_index/GRCh38_gencode_v35" # Path to pre-built STAR index
   GTF_FILE="path/to/your/annotations/gencode.v35.annotation.gtf" # GENCODE v35 GTF file
   NUM_THREADS=8 # Adjust as needed
   READ_LENGTH=150 # Inferred from rMATS parameter --readLength 150
   
   # Create output directory if it doesn't exist
   mkdir -p "${OUTPUT_DIR}/aligned_reads"
   
   # Run STAR alignment
   STAR \
     --genomeDir "${GENOME_DIR}" \
     --readFilesIn "${FASTQ_R1}" "${FASTQ_R2}" \
     --readFilesCommand zcat \
     --runThreadN "${NUM_THREADS}" \
     --outFileNamePrefix "${OUTPUT_DIR}/aligned_reads/${SAMPLE_PREFIX}." \
     --outSAMtype BAM SortedByCoordinate \
     --outSAMunmapped Within \
     --quantMode GeneCounts \
     --sjdbGTFfile "${GTF_FILE}" \
     --sjdbOverhang $((READ_LENGTH - 1)) # Read length - 1 for splice junction database overhang
   

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Tools Used