GSE201898 Processing Pipeline

Publication

Science translational medicine (2022) — PMID 35767654

Dataset

MECP2-related pathways are dysregulated in a cortical organoid model of Myotonic dystrophy

1. 1

   Alignment, cell barcode processing, umis processing, abundance measurements: cellranger count (version 3.0.2)

   Cell Ranger v3.0.2

   $ Bash example

   cellranger_version="3.0.2"
   
   # Cell Ranger is typically installed by downloading the tarball from 10x Genomics and adding it to your PATH.
   # Example (adjust path as needed):
   # wget https://cf.10xgenomics.com/releases/cell-exp/cellranger-3.0.2.tar.gz
   # tar -xzf cellranger-3.0.2.tar.gz
   # export PATH=/path/to/cellranger-3.0.2:$PATH
   
   # Define variables for the run
   SAMPLE_ID="my_sample_id" # A unique ID for this run
   FASTQ_DIR="/path/to/your/fastqs" # Directory containing FASTQ files (e.g., from bcl2fastq or mkfastq)
   SAMPLE_NAME="sample_1" # The sample name prefix for your FASTQ files (e.g., sample_1_S1_L001_R1_001.fastq.gz)
   TRANSCRIPTOME_REF="/path/to/refdata-gex-GRCh38-2020-A" # Path to a Cell Ranger-compatible transcriptome reference (e.g., from 10x Genomics)
   
   # Execute cellranger count
   cellranger count \
       --id=${SAMPLE_ID} \
       --transcriptome=${TRANSCRIPTOME_REF} \
       --fastqs=${FASTQ_DIR} \
       --sample=${SAMPLE_NAME} \
       --expect-cells=3000 # Optional: Expected number of cells, adjust as needed
   

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2. 2

   MD tags were added to alignments with samtools calmd --threads 15 -rb possorted_genome_bam.bam refdata-cellranger-hg19-3.0.0/fasta/genome.fa > possorted_genome_bam_MD.bam

   Cell Ranger v3.0.0 GitHub

   $ Bash example

   # Install samtools if not already available
   # conda install -c bioconda samtools
   
   # Add MD tags to alignments
   samtools calmd --threads 15 -rb possorted_genome_bam.bam refdata-cellranger-hg19-3.0.0/fasta/genome.fa > possorted_genome_bam_MD.bam

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3. 3

   Reads were split based on the CB:Z tag, resulting in one BAM file per barcode.

   fgbio (Inferred with models/gemini-2.5-flash) v2.3.0 GitHub

   $ Bash example

   # Install fgbio if not already installed
   # conda install -c bioconda fgbio
   
   # Define input BAM file (replace with actual input file)
   INPUT_BAM="input.bam"
   
   # Define output prefix for split BAM files
   OUTPUT_PREFIX="barcode_split_"
   
   # Split BAM file based on the CB:Z tag, creating one BAM file per unique barcode.
   # The --tag CB option specifies the tag to split by (CB:Z refers to the CB tag with Z string type).
   # The --strategy BARCODE option ensures splitting by unique barcode values found in the tag.
   fgbio SplitBamByTag --input "${INPUT_BAM}" --output-prefix "${OUTPUT_PREFIX}" --tag CB --strategy BARCODE

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