GSE86479 Processing Pipeline

Publication

Cell metabolism (2017) — PMID 28467932

Dataset

Pseudotemporal ordering of single cells reveals metabolic control of postnatal beta cell proliferation

1. 1

   Reads were aligned to the mm9 by STAR with parameters: --outSAMstrandField intronMotif --outFilterMultimapNmax 1 --runThreadN 5.Only the reads aligned uniquely to one genomic location were retained for subsequent analysis.

   STAR v2.5.x GitHub

   $ Bash example

   # Install STAR (if not already installed)
   # conda install -c bioconda star
   
   # Define variables for reference genome and annotation
   GENOME_DIR="star_mm9_index"
   GENOME_FASTA="mm9.fa"
   GTF_FILE="Mus_musculus.NCBIM37.67.gtf" # Ensembl release 67 for mm9/NCBIM37
   
   # Download reference files (example using curl/wget)
   # mkdir -p ref_data
   # cd ref_data
   # curl -O http://hgdownload.soe.ucsc.edu/goldenPath/mm9/bigZips/mm9.fa.gz
   # gunzip mm9.fa.gz
   # curl -O ftp://ftp.ensembl.org/pub/release-67/gtf/mus_musculus/Mus_musculus.NCBIM37.67.gtf.gz
   # gunzip Mus_musculus.NCBIM37.67.gtf.gz
   # cd ..
   
   # Create STAR genome index (run once per genome)
   # mkdir -p "${GENOME_DIR}"
   # STAR --runMode genomeGenerate \
   #      --genomeDir "${GENOME_DIR}" \
   #      --genomeFastaFiles "ref_data/${GENOME_FASTA}" \
   #      --sjdbGTFfile "ref_data/${GTF_FILE}" \
   #      --sjdbOverhang 100 \
   #      --runThreadN 5 # Use the same number of threads as alignment if possible
   
   # Define input and output files
   READ1="sample_R1.fastq.gz"
   READ2="sample_R2.fastq.gz" # Assuming paired-end reads
   OUTPUT_PREFIX="aligned_reads/"
   
   # Create output directory
   mkdir -p "${OUTPUT_PREFIX}"
   
   # Align reads with STAR
   STAR --genomeDir "${GENOME_DIR}" \
        --readFilesIn "${READ1}" "${READ2}" \
        --readFilesCommand zcat \
        --outFileNamePrefix "${OUTPUT_PREFIX}" \
        --runThreadN 5 \
        --outSAMstrandField intronMotif \
        --outFilterMultimapNmax 1 \
        --outSAMtype BAM SortedByCoordinate \
        --outSAMattributes NH HI AS NM MD \
        --outSAMmapqUnique 60 \
        --outFilterType BySJout \
        --outFilterMismatchNmax 999 \
        --outFilterMismatchNoverLmax 0.04 \
        --alignIntronMin 20 \
        --alignIntronMax 1000000 \
        --alignMatesGapMax 1000000 \
        --limitBAMsortRAM 30000000000 # Adjust based on available RAM (e.g., 30GB)

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2. 2

   makeTagDirectory and makeUCSCfile in homer was used to generate bigWig file

   HOMER vInferred with models/gemini-2.5-flash GitHub

   $ Bash example

   # Install HOMER (if not already installed)
   # conda install -c bioconda homer
   
   # Define variables (replace with actual paths and names)
   INPUT_BAM="input_sample.bam"
   TAG_DIRECTORY="sample_tag_directory"
   OUTPUT_BIGWIG="sample_signal.bigWig"
   GENOME_ASSEMBLY="hg38" # Placeholder: Replace with your reference genome (e.g., mm10, hg38)
   
   # Step 1: Create a Tag Directory from BAM/SAM files
   # This command processes the alignment file(s) and creates a directory containing tag information.
   # -format bam: Specifies the input file format.
   # -genome: Specifies the reference genome assembly for proper normalization and coordinate handling.
   # -tbp 1: Treats each read as a single tag (useful for single-end reads or when fragment length is handled later).
   # -fragLength given: Tells HOMER to use the fragment length specified in the BAM file (if paired-end) or infer it.
   makeTagDirectory ${TAG_DIRECTORY} ${INPUT_BAM} -format bam -genome ${GENOME_ASSEMBLY} -tbp 1 -fragLength given
   
   # Step 2: Generate a bigWig file from the Tag Directory
   # This command converts the tag directory into a bigWig file suitable for visualization in genome browsers.
   # -o: Specifies the output bigWig file name.
   # -bigWig: Ensures the output is in bigWig format.
   # -norm 1x: Normalizes the signal to 1x coverage (reads per million mapped reads), a common method for visualization.
   # -res 10: Sets the resolution of the bigWig file to 10 bp.
   makeUCSCfile ${TAG_DIRECTORY} -o ${OUTPUT_BIGWIG} -bigWig -norm 1x -res 10

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Tools Used