GSE133479 Processing Pipeline

Publication

Nature communications (2020) — PMID 31992716

Dataset

Cancer avatars derived from genetically engineered pluripotent stem cells allow for longitudinal assessment of tumor development

1. 1

   RNA-seq reads were aligned to the human genome (hg19) with STAR 2.4.0h (outFilterMultimapNmax 20, outFilterMismatchNmax 999, outFilterMismatchNoverLmax 0.04, outFilterIntronMotifs RemoveNoncanonicalUnannotated, outSJfilterOverhangMin 6 6 6 6, seedSearchStartLmax 20, alignSJDBoverhangMin 1) using a gene database constructed from Gencode v19

   STAR v2.4.0h GitHub

   $ Bash example

   # Install STAR (if not already installed)
   # conda install -c bioconda star
   
   # Define variables
   READ1_FASTQ="input_R1.fastq.gz" # Replace with your R1 fastq file
   # READ2_FASTQ="input_R2.fastq.gz" # Uncomment and replace if paired-end
   OUTPUT_PREFIX="aligned_reads/sample_name" # Replace with your desired output prefix
   STAR_INDEX_DIR="/path/to/your/star_index/hg19_gencode_v19" # Replace with the path to your STAR index (built with hg19 and Gencode v19)
   NUM_THREADS=8 # Adjust as needed
   
   # Create output directory if it doesn't exist
   mkdir -p $(dirname "${OUTPUT_PREFIX}")
   
   # Run STAR alignment
   STAR \
     --runThreadN "${NUM_THREADS}" \
     --genomeDir "${STAR_INDEX_DIR}" \
     --readFilesIn "${READ1_FASTQ}" \
     # --readFilesIn "${READ1_FASTQ}" "${READ2_FASTQ}" # Uncomment if paired-end
     --readFilesCommand zcat \
     --outFileNamePrefix "${OUTPUT_PREFIX}" \
     --outFilterMultimapNmax 20 \
     --outFilterMismatchNmax 999 \
     --outFilterMismatchNoverLmax 0.04 \
     --outFilterIntronMotifs RemoveNoncanonicalUnannotated \
     --outSJfilterOverhangMin 6 6 6 6 \
     --seedSearchStartLmax 20 \
     --alignSJDBoverhangMin 1 \
     --outSAMtype BAM SortedByCoordinate \
     --outSAMunmapped Within \
     --outSAMattributes All \
     --outSAMstrandField intronMotif

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2. 2

   Reads that overlap with exon coordinates were counted using HTSeqcount (-s reverse -a 0 -t exon -i gene_id -m union)

   HTSeq (Inferred with models/gemini-2.5-flash) v0.13.5 (Inferred with models/gemini-2.5-flash) GitHub

   $ Bash example

   # Install HTSeq (if not already installed)
   # conda install -c bioconda htseq
   
   # Placeholder for aligned reads (BAM/SAM) and annotation (GTF/GFF)
   # Replace 'aligned_reads.bam' with your actual aligned reads file.
   # Replace 'annotation.gtf' with your actual gene annotation file (e.g., from GENCODE, Ensembl).
   # Ensure the GTF/GFF file contains 'exon' features and 'gene_id' attributes.
   
   htseq-count -s reverse -a 0 -t exon -i gene_id -m union aligned_reads.bam annotation.gtf > gene_counts.txt

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Tools Used