ISO 20397-2:2021

Overview

ISO 20397-2:2021 - "Biotechnology - Massively parallel sequencing - Part 2: Quality evaluation of sequencing data" provides general requirements and recommendations for assessing and controlling the quality of massively parallel sequencing (MPS) data after raw data generation. The standard focuses on post-sequencing processes including raw data quality assessment, sequence alignment/mapping, variant calling, validation, and documentation. It explicitly does not apply to de novo assembly workflows.

Keywords: ISO 20397-2, massively parallel sequencing (MPS), sequencing data quality, quality evaluation, variant calling, sequence alignment.

Key Topics and Requirements

• Raw data quality
  • Definitions of raw data and recommended metrics for assessing base calling, base quality, read length, GC content, duplication level and adapter contamination.
  • Guidance on raw data pre-processing (trimming, demultiplexing) and basic statistics to monitor.
• Sequence alignment and mapping
  • Expectations for alignment/mapping file formats and quality control metrics (mapping rate, coverage depth/breadth, alignment quality indicators).
  • Recommendations for alignment post-processing and methods to evaluate mapping quality.
• Variant calling
  • Requirements for input data files, variant-calling quality metrics, handling of false positives, and sequence annotation considerations.
• Validation and documentation
  • General guidelines for validating quality metrics and documenting the pipeline, parameters and results to support reproducibility and traceability.
• Annexes
  • Informative annexes provide platform-specific quality metrics, coverage/read recommendations for applications, and a non-exhaustive list of alignment/mapping software.

Applications and Who Uses It

ISO 20397-2 is applicable to organizations and professionals involved in MPS data generation and analysis, including:

• Clinical and diagnostic laboratories validating sequencing-based tests
• Genomics and biotech companies running high-throughput sequencing
• Core sequencing facilities and service providers
• Bioinformaticians designing and validating pipelines for alignment and variant calling
• Quality managers and regulatory bodies requiring documented quality control and traceability

Practical benefits include improved data reliability, consistent quality monitoring across pipelines, and clearer documentation for compliance, research reproducibility, and clinical validation.

Related Standards

• Other parts of the ISO 20397 series (see ISO website for the full list)
• Referenced terminology sources used in the standard (e.g., ISO online browsing platform)

This standard is a practical resource for organizations seeking structured, standardized guidance on MPS quality evaluation, enabling robust downstream analyses such as accurate variant detection and reproducible genomics workflows.