5. Read Trimming, Adapter Clipping & Filtering with fastp

Residual sequencing adapters, low-quality ends, and two-color optical noise (e.g., NovaSeq poly-G tails where no fluorophore fires) compromise spliced alignment accuracy. fastp is an ultra-fast C++ preprocessor that automatically detects adapters, clips sliding-window low quality bases, trims poly-G, and outputs interactive HTML diagnostics in a single pass.

Automated Batch Preprocessing with fastp

# Loop over all samples and execute fastp trimming
for R1 in 00_raw_data/*_R1.fastq.gz; do
    SAMPLE=$(basename "$R1" _R1.fastq.gz)
    R2="00_raw_data/${SAMPLE}_R2.fastq.gz"

    fastp \
      --in1 "$R1" \
      --in2 "$R2" \
      --out1 "02_trimmed_data/${SAMPLE}_R1.trimmed.fastq.gz" \
      --out2 "02_trimmed_data/${SAMPLE}_R2.trimmed.fastq.gz" \
      --detect_adapter_for_pe \
      --trim_poly_g \
      --cut_front --cut_tail \
      --cut_mean_quality 20 \
      --length_required 36 \
      --thread 8 \
      --html "03_qc_trimmed/${SAMPLE}_fastp.html" \
      --json "03_qc_trimmed/${SAMPLE}_fastp.json"
done

# Generate post-trimming MultiQC report
multiqc 03_qc_trimmed/ -o 03_qc_trimmed/ --title "Post-Trimming MultiQC Report"