GSE89036 Processing Pipeline

Publication

Nature immunology (2017) — PMID 28218746

Dataset

Genome-wide maps of H3K27me3 in CD8 T cell subsets

1. 1

   Reads were aligned to mm10 by BWA

   BWA v0.7.17 GitHub

   $ Bash example

   # Install BWA and Samtools if not already installed
   # conda install -c bioconda bwa samtools
   
   # --- Reference Genome Preparation ---
   # Download the mm10 reference genome (example from UCSC)
   # mkdir -p ref/mm10
   # wget -P ref/mm10 https://hgdownload.soe.ucsc.edu/goldenPath/mm10/bigZips/mm10.fa.gz
   # gunzip ref/mm10/mm10.fa.gz
   
   # Index the reference genome for BWA
   # bwa index ref/mm10/mm10.fa
   
   # --- Alignment Command ---
   # Assuming input reads are paired-end: reads_R1.fastq.gz and reads_R2.fastq.gz
   # And the reference genome is indexed at ref/mm10/mm10.fa
   bwa mem -t 8 ref/mm10/mm10.fa reads_R1.fastq.gz reads_R2.fastq.gz | samtools view -bS -o aligned_reads.bam -

   Copy View on GitHub
2. 2

   Reads with low quality (MAPQ < 30) were filtered out.

   samtools (Inferred with models/gemini-2.5-flash) v1.10 (Inferred with models/gemini-2.5-flash) GitHub

   $ Bash example

   # Install samtools (if not already installed)
   # conda install -c bioconda samtools
   
   # Filter reads with mapping quality (MAPQ) less than 30
   samtools view -b -q 30 -o filtered_reads.bam input_reads.bam

   Copy View on GitHub
3. 3

   If multiple reads were mapped to the exact same location, only one read was kept.

   samtools markdup (Inferred with models/gemini-2.5-flash) v1.15 GitHub

   $ Bash example

   # Install samtools if not already installed
   # conda install -c bioconda samtools=1.15
   
   # Input: A coordinate-sorted BAM file. This step assumes the input BAM is already sorted.
   # Example input from a previous alignment step:
   INPUT_BAM="aligned_reads.sorted.bam"
   OUTPUT_DEDUP_BAM="aligned_reads.dedup.bam"
   
   # Remove PCR duplicates based on mapping coordinates.
   # If multiple reads map to the exact same genomic location, only one is kept.
   # -r: Remove duplicate reads (rather than just marking them).
   # -s: Output statistics to stderr (optional, but useful for logging).
   samtools markdup -r -s "${INPUT_BAM}" "${OUTPUT_DEDUP_BAM}"

   Copy View on GitHub