ngs-read-preprocessing

Skill: ngs-read-preprocessing

Use When

• The user wants to trim adapters from raw sequencing reads.
• The user needs to filter low-quality reads or bases before downstream alignment.
• The user wants to generate clean FASTQ files suitable for alignment or assembly.
• The user needs a per-sample HTML QC report showing before/after statistics.
• The user wants to perform polyG or polyX tail trimming for NextSeq/NovaSeq platforms.
• The user needs to apply minimum read length filtering after trimming.

Inputs

• Required:
  • Input FASTQ R1 (-i FILE) — single-end read file or forward reads for paired-end.
• Optional:
  • Input FASTQ R2 (-I FILE) — reverse reads for paired-end sequencing.
  • --outdir DIR — Output directory (default: fastp_results).
  • --threads N — Number of worker threads (default: 4).
  • --min-qual N — Minimum qualified quality phred value for sliding window filtering (default: 15).
  • --min-len N — Minimum read length after trimming (default: 36 bp).
  • --polyg — Enable polyG tail trimming (recommended for NextSeq/NovaSeq two-color chemistry).
  • Adapter sequences — auto-detected by default; user may supply explicit sequences via additional fastp flags.
  • Additional fastp flags passed through as extra arguments.

Workflow

1. Validate that input FASTQ files exist and are readable.
2. Detect single-end vs. paired-end mode from the provided inputs (-i only vs. -i and -I).
3. Run fastp with adapter auto-detection (or user-specified adapter sequences if provided).
4. Apply quality filtering using a sliding window approach (default: Q15 qualified quality threshold).
5. Apply length filtering to discard reads shorter than the minimum length (default: 36 bp).
6. Enable polyG tail trimming for NextSeq/NovaSeq platforms if --polyg is specified.
7. Generate an HTML report and JSON statistics file with detailed before/after metrics.
8. Report summary: reads before/after filtering, bases before/after, Q20/Q30 rates, and adapter trimming rate.

Output Contract

• Cleaned FASTQ file(s) — Trimmed and filtered reads (<sample>_trimmed.fastq.gz; R1 and R2 for paired-end).
• fastp HTML report — Per-sample visual QC summary (<sample>_fastp.html).
• fastp JSON report — Machine-readable statistics (<sample>_fastp.json).
• Summary statistics — Reads in/out, bases in/out, Q20/Q30 rates before and after filtering, adapter detection/trimming rate.

Limits

• fastp must be installed and available on $PATH.
• fastp is single-threaded by default; use -w / --threads to enable multi-threading (up to 16 threads).
• This skill does not perform read alignment; use genome-read-alignment or rnaseq-read-alignment for that purpose.
• This skill does not aggregate QC across multiple samples; use ngs-quality-control for multi-sample QC aggregation with MultiQC.
• UMI processing is supported by fastp but is not enabled by default; pass --umi and related flags as extra arguments if needed.
• Interleaved FASTQ input is not supported by the wrapper script; use fastp directly for interleaved input.
• Common failure cases:
  • Paired-end FASTQ files with mismatched read counts causing fastp to abort.
  • Incorrect adapter sequences provided, leading to untrimmed reads.
  • Disk full during output writing, producing truncated FASTQ files.