GSE83687 Processing Pipeline

Publication

Gut (2022) — PMID 34853057

Dataset

A functional genomics predictive network model identifies regulators of inflammatory bowel disease: Mount Sinai Hospital (MSH) Population Specimen Co…

1. 1

   Illumina Casava1.82 software used for basecalling.

   Illumina Casava v1.82

   $ Bash example

   # The description states "Illumina Casava1.82 software used for basecalling."
   # Casava was an integrated software suite for processing Illumina sequencing data,
   # including basecalling (converting BCL files to FASTQ).
   # The specific command-line execution for Casava 1.8.2 is not provided in the description.
   # Basecalling is the process of converting raw intensity data (BCL files) into base calls and quality scores, typically outputting FASTQ files.
   # Modern Illumina basecalling is typically performed using bcl2fastq or bcl-convert.
   # No specific parameters or reference datasets are mentioned in the description.

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2. 2

   Short reads in fastQ format are processed using RAPiD, which is a RNA-Seq analysis framework developed and maintained by the Technology Development group at Icahn Institute for Genomics and MultiScale Biology. .

   RNA-seq vNot specified (Inferred with models/gemini-2.5-flash)

   $ Bash example

   # RAPiD is described as an RNA-Seq analysis framework developed and maintained by the Technology Development group at Icahn Institute for Genomics and MultiScale Biology.
   # As an internal framework, specific installation instructions and command-line interface details are not publicly available.
   # The following is a conceptual representation of how such a tool would be invoked, assuming standard RNA-Seq inputs.
   
   # Placeholder for input FASTQ files
   # For single-end reads:
   # INPUT_FASTQ="sample_reads.fastq.gz"
   # For paired-end reads:
   # INPUT_FASTQ_R1="sample_R1.fastq.gz"
   # INPUT_FASTQ_R2="sample_R2.fastq.gz"
   
   # Placeholder for reference genome and annotation files (e.g., human hg38)
   # GENOME_FASTA="/path/to/reference/GRCh38.p14.genome.fa"
   # GENE_ANNOTATION_GTF="/path/to/reference/gencode.v45.annotation.gtf"
   
   # Placeholder for output directory
   # OUTPUT_DIR="rapid_analysis_output"
   
   # Conceptual command for running RAPiD. The actual command would depend on the framework's design.
   # rapid_run --fastq $INPUT_FASTQ --genome $GENOME_FASTA --gtf $GENE_ANNOTATION_GTF --output $OUTPUT_DIR
   # rapid_run --fastq-r1 $INPUT_FASTQ_R1 --fastq-r2 $INPUT_FASTQ_R2 --genome $GENOME_FASTA --gtf $GENE_ANNOTATION_GTF --output $OUTPUT_DIR
   
   echo "RAPiD is an internal RNA-Seq analysis framework. Specific command-line usage and parameters are not publicly documented."
   echo "Please consult the Technology Development group at Icahn Institute for Genomics and MultiScale Biology for detailed instructions."

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3. 3

   RAPiD uses STAR to map the short reads to the [hg19 ] reference and resultant alignment map in BAM format is quantified for gene level expression using featureCounts of the subreads package.

   STAR v2.7.10a GitHub

   $ Bash example

   # Install STAR (if not already installed)
   # conda install -c bioconda star
   
   # Define variables
   # Placeholder for hg19 STAR index. This directory should contain genome files (e.g., genome.fa, SA, SAindex, etc.)
   GENOME_DIR="/path/to/STAR_index/hg19"
   # Placeholder for input R1 FASTQ file (e.g., sample_R1.fastq.gz)
   READS_R1="input_reads_R1.fastq.gz"
   # Placeholder for input R2 FASTQ file (e.g., sample_R2.fastq.gz). Remove if single-end.
   READS_R2="input_reads_R2.fastq.gz"
   # Prefix for output files (e.g., sample_aligned.bam)
   OUTPUT_PREFIX="sample_aligned"
   # Number of threads to use for alignment
   THREADS=8
   
   # Run STAR alignment
   # --genomeDir: Path to the STAR genome index directory
   # --readFilesIn: Input FASTQ files (space-separated for paired-end)
   # --runThreadN: Number of threads
   # --outFileNamePrefix: Prefix for all output files
   # --outSAMtype BAM SortedByCoordinate: Output sorted BAM file
   # --readFilesCommand zcat: Command to decompress gzipped FASTQ files
   STAR --genomeDir "${GENOME_DIR}" \
        --readFilesIn "${READS_R1}" "${READS_R2}" \
        --runThreadN "${THREADS}" \
        --outFileNamePrefix "${OUTPUT_PREFIX}" \
        --outSAMtype BAM SortedByCoordinate \
        --readFilesCommand zcat

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4. 4

   Detailed QC metrics are generated using the RNASeQC package

   RNASeQC vv2.3.6 (Inferred with models/gemini-2.5-flash) GitHub

   $ Bash example

   # Install RNASeQC via conda
   # conda install -c bioconda rnaseqc
   
   # Define variables for input and output
   INPUT_BAM="sample_aligned.bam" # Placeholder: Replace with actual path to input BAM file
   REFERENCE_FASTA="GRCh38.primary_assembly.genome.fa" # Placeholder: Replace with actual path to reference genome FASTA (e.g., from GENCODE or Ensembl)
   GENE_MODEL_GTF="gencode.v45.annotation.gtf" # Placeholder: Replace with actual path to gene model GTF (e.g., from GENCODE)
   OUTPUT_DIR="rnaseqc_output"
   SAMPLE_ID="sample1" # Identifier for the sample, used in reports
   
   # Create output directory if it doesn't exist
   mkdir -p "${OUTPUT_DIR}"
   
   # Execute RNA-SeQC
   # The 'rnaseqc' command from bioconda is a wrapper that typically calls the Java JAR.
   # The arguments are consistent with the direct Java command.
   rnaseqc \
     -o "${OUTPUT_DIR}" \
     -r "${REFERENCE_FASTA}" \
     -g "${GENE_MODEL_GTF}" \
     -s "${SAMPLE_ID}|${INPUT_BAM}|RNA" # Format: SampleID|PathToBam|SampleType (e.g., RNA, DNA)

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Tools Used