GSE185373 Processing Pipeline

Publication

Molecular cell (2023) — PMID 37084731

Dataset

Function-first proteomic strategies for chemical probe discovery in human cells

1. 1

   FASTQ files were first trimmed using Trim_galore (v0.6.4) to remove sequencing adapters and low quality (Q<15) reads.

   Trim Galore v0.6.4 GitHub

   $ Bash example

   # Install Trim Galore (and its dependencies like Cutadapt and FastQC)
   # conda install -c bioconda trim-galore
   
   # Define input and output paths
   INPUT_FASTQ="input.fastq.gz" # Placeholder for your input FASTQ file
   OUTPUT_DIR="trimmed_fastq"
   
   # Create output directory if it doesn't exist
   mkdir -p "${OUTPUT_DIR}"
   
   # Run Trim Galore to remove sequencing adapters and low quality (Q<15) reads
   # --quality 15: Trims reads from the 3' end until the average quality in a window is above 15
   # --output_dir: Specifies the directory for output files
   # Trim Galore automatically detects and removes common sequencing adapters.
   # For paired-end data, use: trim_galore --paired --quality 15 --output_dir "${OUTPUT_DIR}" input_R1.fastq.gz input_R2.fastq.gz
   trim_galore --quality 15 --output_dir "${OUTPUT_DIR}" "${INPUT_FASTQ}"

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2. 2

   Trimmed sequencing reads were aligned to the human Hg19 reference genome (GENCODE, GRCh37.p13) using STAR (v2.7.5).

   STAR v2.7.5 GitHub

   $ Bash example

   # Install STAR (if not already installed)
   # conda install -c bioconda star=2.7.5
   
   # Define variables
   STAR_VERSION="2.7.5"
   # Placeholder for the STAR genome index for human Hg19 (GRCh37.p13) with GENCODE annotations.
   # This index would typically be pre-built using a command like:
   # STAR --runThreadN <threads> --runMode genomeGenerate \
   #      --genomeDir /path/to/STAR_index_hg19_gencode \
   #      --genomeFastaFiles /path/to/GRCh37.p13.genome.fa \
   #      --sjdbGTFfile /path/to/gencode.v19.annotation.gtf \
   #      --sjdbOverhang 100 # Adjust based on read length
   GENOME_DIR="/path/to/STAR_index_hg19_gencode_GRCh37.p13"
   READS_FILE="trimmed_reads.fastq.gz" # Placeholder for input trimmed reads
   OUTPUT_PREFIX="aligned_reads"
   THREADS=8 # A common default for --runThreadN
   
   # Align reads using STAR
   STAR --runThreadN ${THREADS} \
        --genomeDir ${GENOME_DIR} \
        --readFilesIn ${READS_FILE} \
        --readFilesCommand zcat \
        --outFileNamePrefix ${OUTPUT_PREFIX}_ \
        --outSAMtype BAM SortedByCoordinate \
        --outSAMunmapped Within \
        --outSAMattributes All

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3. 3

   SAM files were subsequently converted to BAM files, sorted, and indexed using samtools (v1.9).

   samtools v1.9 GitHub

   $ Bash example

   # Install samtools if not already installed
   # conda install -c bioconda samtools=1.9
   
   # Placeholder for input SAM file
   INPUT_SAM="input.sam"
   # Placeholder for output sorted BAM file
   OUTPUT_BAM_SORTED="output_sorted.bam"
   
   # Convert SAM to BAM and sort in one step
   # samtools sort can take SAM as input and output a sorted BAM file
   samtools sort "${INPUT_SAM}" -o "${OUTPUT_BAM_SORTED}"
   
   # Index the sorted BAM file
   samtools index "${OUTPUT_BAM_SORTED}"

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4. 4

   BAM files were used to generate bigwig files using bamCoverage (part of the Deeptools package; v3.3.1).

   deepTools v3.3.1 GitHub

   $ Bash example

   # Install deepTools (if not already installed)
   # conda install -c bioconda deeptools=3.3.1
   
   # Example usage: Generate a bigwig file from a BAM file
   # Replace 'input.bam' with your actual BAM file path
   # Replace 'output.bw' with your desired output bigwig file path
   bamCoverage -b input.bam -o output.bw --binSize 10 --numberOfProcessors auto

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Tools Used