GSE135300 Processing Pipeline

Publication

Nature cancer (2020) — PMID 34109316

Dataset

Next Generation Sequencing: in vivo genome-wide CRISPR sgRNA screen in primary cancer-initiating and propagating bcCML stem cells

1. 1

   Reads were counted by first searching for the CACCG sequence in the primary read file that appears in the vector 5’ to all sgRNA inserts.

   grep (Inferred with models/gemini-2.5-flash) vN/A

   $ Bash example

   # This command counts the number of lines in the primary read file that contain the sequence "CACCG".
   # The '-c' option tells grep to output only a count of matching lines.
   # Replace 'primary_read_file.fastq' with the actual path to your read file.
   grep -c "CACCG" primary_read_file.fastq > cac_sequence_count.txt

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2. 2

   The next 20 nts are the sgRNA insert, which was then mapped to a reference file of all possible sgRNAs present in the library.

   bowtie2 (Inferred with models/gemini-2.5-flash) v2.4.5 GitHub

   $ Bash example

   # Install bowtie2 if not already installed
   # conda install -c bioconda bowtie2
   
   # Define input and output files
   # Assuming 'sgRNA_reads.fastq' contains the extracted 20nt sgRNA inserts
   SGRNA_READS="sgRNA_reads.fastq"
   # 'sgRNA_library.fasta' is the reference file of all possible sgRNAs
   SGRNA_LIBRARY_FASTA="sgRNA_library.fasta"
   INDEX_PREFIX="sgRNA_library_index"
   OUTPUT_SAM="sgRNA_mapped.sam"
   THREADS=8 # Example number of threads
   
   # 1. Build Bowtie2 index for the sgRNA library
   # This step only needs to be run once for a given reference library
   bowtie2-build "${SGRNA_LIBRARY_FASTA}" "${INDEX_PREFIX}"
   
   # 2. Map the sgRNA inserts to the indexed library
   # -U for single-end reads
   # -S for SAM output
   # -p for number of threads
   bowtie2 -x "${INDEX_PREFIX}" -U "${SGRNA_READS}" -S "${OUTPUT_SAM}" -p "${THREADS}"
   
   # Optional: Convert SAM to BAM, sort, and index
   # samtools view -bS "${OUTPUT_SAM}" > "${OUTPUT_SAM%.sam}.bam"
   # samtools sort "${OUTPUT_SAM%.sam}.bam" -o "${OUTPUT_SAM%.sam}.sorted.bam"
   # samtools index "${OUTPUT_SAM%.sam}.sorted.bam"

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