National foreword

This British Standard is the UK implementation of ISO 20397‑3:2025.

The UK participation in its preparation was entrusted to Technical Committee BTI/1, Biotechnologies.

A list of organizations represented on this committee can be obtained on request to its committee manager.

Contractual and legal considerations

This publication has been prepared in good faith, however no representation, warranty, assurance or undertaking (express or implied) is or will be made, and no responsibility or liability is or will be accepted by BSI in relation to the adequacy, accuracy, completeness or reasonableness of this publication. All and any such responsibility and liability is expressly disclaimed to the full extent permitted by the law.

This publication is provided as is, and is to be used at the recipient’s own risk.

The recipient is advised to consider seeking professional guidance with respect to its use of this publication.

This publication is not intended to constitute a contract. Users are responsible for its correct application.

Compliance with a British Standard cannot confer immunity from legal obligations.

This British Standard was published under the authority of the Standards Policy and Strategy Committee on 31 July 2025.

Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the different types of ISO document should be noted. This document was drafted in accordance with the editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).

ISO draws attention to the possibility that the implementation of this document may involve the use of (a) patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a) patent(s) which may be required to implement this document. However, implementers are cautioned that this may not represent the latest information, which may be obtained from the patent database available at www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.

Any trade name used in this document is information given for the convenience of users and does not constitute an endorsement.

For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and expressions related to conformity assessment, as well as information about ISO's adherence to the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/iso/foreword.html.

This document was prepared by Technical Committee ISO/TC 276 Biotechnology, Subcommittee SC 1, Analytical methods.

A list of all parts in the ISO 20397 series can be found on the ISO website.

Any feedback or questions on this document should be directed to the user’s national standards body. A complete listing of these bodies can be found at www.iso.org/members.html.

Introduction

Massively parallel sequencing (MPS) is a high-throughput analytical approach to nucleic acid sequencing utilizing massively parallel processing, that allows whole genomes, transcriptomes, and specific nucleic acid targets from different organisms to be investigated in a relatively short time.

Metagenomics approaches are an extremely powerful strategy in large-scale genomics applications as a way to study the taxonomic and functional composition of microbial communities from environmental, agricultural, and clinical primary samples/samples. Metagenomics does not require isolation of single bacterium from complex microbial community, but catalogues by sequencing all genes and genomes from total DNA (tDNA). It has great advantages to identify new species, including microorganisms that are difficult to culture under typical laboratory conditions.

Analysing metagenomics data involves a complex and statistically driven process that extends beyond traditional MPS pipelines to include identification, functional and relative abundance analyses. In metagenomics, whole genomic DNA is prepared from primary samples, regardless of its microbial composition and is characterized by whole genome sequencing. The annotation of resulting DNA fragments, individual reads or assembled sequence contigs, to individual taxonomic groups or known genome sequences, is carried out by sophisticated bioinformatic tools. The analysis is not limited to traditional MPS pipeline but also focuses on the identification of functional composition of a microbial community, which include the assignment of protein-coding open reading frames to functional categories, such as protein domain families or gene ontologies. Consequently, the analysis of whole genome sequencing (WGS) metagenomics data sets involves a significant statistical component, as sequence data must be evaluated based on relative abundances rather than on absolute presence/absence data.

1 Scope

This document specifies general requirements and guidance for metagenomics-dedicated sample preparation, and generating and analysing metagenomics sequence data obtained from massive parallel sequencing platforms. The specified metagenomics process includes the following stages:

sampling strategy and process, including type, storage, transportation, extraction, quality;
nucleic acid library preparation
design and review process including sequencing strategy and assessment;
database construction;
bioinformatics analysis and report
validation and verification for bioinformatics pipeline, and database

This document applies to laboratories and research organizations.

2 Normative references

3 Terms and definitions

4 Principle

4.1 General

5 Sampling strategy

5.1 General

5.2 Primary sample type

5.3 Primary sample stabilization and storage

5.4 Primary sample transportation

5.5 DNA/RNA isolation

5.6 DNA/RNA sample quality

6 Nucleic acid library preparation

7 Design and review process including sequencing strategy and assessment

7.1 General

7.2 Short read

7.3 Long read

7.4 Hybrid assembly

7.5 Sample preparation and library construction

7.6 Assessment

8 Database construction

8.1 General

8.2 Public database

8.3 Self-build database

9 Bioinformatics analysis

9.1 General

9.2 Identification list of microorganisms

10 Validation and verification

10.1 General

10.2 In silico sequence control for bioinformatics pipeline evaluation

10.3 Real sample control for pipeline evaluation

11 Evaluation

12 Test report

12.1 General

12.2 Test report content

Bibliography

BS ISO 20397-3:2025

Biotechnology. Massively parallel sequencing - General requirements and guidance for metagenomics

Current

Published:

31 Jul 2025