ISO 20397-3:2025

Biotechnology — Massively parallel sequencing — Part 3: General requirements and guidance for metagenomics

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: a) sampling strategy and process, including type, storage, transportation, extraction, quality; b) nucleic acid library preparation c) design and review process including sequencing strategy and assessment; d) database construction; e) bioinformatics analysis and report f) validation and verification for bioinformatics pipeline, and database This document applies to laboratories and research organizations.

Biotechnologie — Séquençage à grande échelle — Partie 3: Exigences générales et recommandations pour la métagénomique

General Information

• Status: Published
• Publication Date: 22-Jul-2025
• ICS: 07.080 - Biology. Botany. Zoology
• Technical Committee: ISO/TC 276 - Biotechnology
• Drafting Committee: ISO/TC 276 - Biotechnology
• Current Stage: 6060 - International Standard published
• Start Date: 23-Jul-2025
• Due Date: 05-Jul-2025
• Completion Date: 23-Jul-2025

Standards Content (Sample)

International
Standard
ISO 20397-3
First edition
Biotechnology — Massively parallel
2025-07
sequencing —
Part 3:
General requirements and guidance
for metagenomics
Biotechnologie — Séquençage à grande échelle —
Partie 3: Exigences générales et recommandations pour la
métagénomique
Reference number
© ISO 2025
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 4
4.1 General .4
5 Sampling strategy . 5
5.1 General .5
5.2 Primary sample type .6
5.3 Primary sample stabilization and storage .6
5.4 Primary sample transportation .6
5.5 DNA/RNA isolation .7
5.6 DNA/RNA sample quality .7
6 Nucleic acid library preparation . 7
7 Design and review process including sequencing strategy and assessment . 7
7.1 General .7
7.2 Short read . .7
7.3 Long read .8
7.4 Hybrid assembly .8
7.5 Sample preparation and library construction.8
7.6 Assessment .8
8 Database construction . 9
8.1 General .9
8.2 Public database .9
8.3 Self-build database .9
9 Bioinformatics analysis . 10
9.1 General .10
9.2 Identification list of microorganisms .11
10 Validation and verification .11
10.1 General .11
10.2 In silico sequence control for bioinformatics pipeline evaluation.11
10.3 Real sample control for pipeline evaluation .11
11 Evaluation .12
12 Test report .12
12.1 General . 12
12.2 Test report content . 12
Annex A (informative) Checklist for NA sample quality assessment before library construction .13
Annex B (informative) Methods for sample stabilization and storage . 14
Annex C (informative) Bioinformatics pipeline .15
Bibliography . 17

iii
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.

iv
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.

v
International Standard ISO 20397-3:2025(en)
Biotechnology — Massively parallel sequencing —
Part 3:
General requirements and guidance for metagenomics
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 p
...

FINAL DRAFT
International
Standard
ISO/FDIS 20397-3
ISO/TC 276/SC 1
Biotechnology — Massively parallel
Secretariat: ANSI
sequencing —
Voting begins on:
2025-04-30
Part 3:
General requirements and guidance
Voting terminates on:
2025-06-25
for metagenomics
RECIPIENTS OF THIS DRAFT ARE INVITED TO SUBMIT,
WITH THEIR COMMENTS, NOTIFICATION OF ANY
RELEVANT PATENT RIGHTS OF WHICH THEY ARE AWARE
AND TO PROVIDE SUPPOR TING DOCUMENTATION.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO-
LOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT
INTERNATIONAL STANDARDS MAY ON OCCASION HAVE
TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL
TO BECOME STAN DARDS TO WHICH REFERENCE MAY BE
MADE IN NATIONAL REGULATIONS.
Reference number
ISO/FDIS 20397-3:2025(en) © ISO 2025

FINAL DRAFT
ISO/FDIS 20397-3:2025(en)
International
Standard
ISO/FDIS 20397-3
ISO/TC 276/SC 1
Biotechnology — Massively parallel
Secretariat: ANSI
sequencing —
Voting begins on:
Part 3:
General requirements and guidance
Voting terminates on:
for metagenomics
RECIPIENTS OF THIS DRAFT ARE INVITED TO SUBMIT,
WITH THEIR COMMENTS, NOTIFICATION OF ANY
RELEVANT PATENT RIGHTS OF WHICH THEY ARE AWARE
AND TO PROVIDE SUPPOR TING DOCUMENTATION.
© ISO 2025
IN ADDITION TO THEIR EVALUATION AS
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO-
LOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
INTERNATIONAL STANDARDS MAY ON OCCASION HAVE
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL
or ISO’s member body in the country of the requester.
TO BECOME STAN DARDS TO WHICH REFERENCE MAY BE
MADE IN NATIONAL REGULATIONS.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland Reference number
ISO/FDIS 20397-3:2025(en) © ISO 2025

ii
ISO/FDIS 20397-3:2025(en)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 4
4.1 General .4
5 Sampling strategy . 5
5.1 General .5
5.2 Primary sample type .6
5.3 Primary sample stabilization and storage .6
5.4 Primary sample transportation .6
5.5 DNA/RNA isolation .7
5.6 DNA/RNA sample quality .7
6 Nucleic acid library preparation . 7
7 Design and review process including sequencing strategy and assessment . 7
7.1 General .7
7.2 Short read . .7
7.3 Long read .8
7.4 Hybrid assembly .8
7.5 Sample preparation and library construction.8
7.6 Assessment .8
8 Database construction . 9
8.1 General .9
8.2 Public database .9
8.3 Self-build database .9
9 Bioinformatics analysis . 10
9.1 General .10
9.2 Identification list of microorganisms .11
10 Validation and verification .11
10.1 General .11
10.2 In silico sequence control for bioinformatics pipeline evaluation.11
10.3 Real sample control for pipeline evaluation .11
11 Evaluation .12
12 Test report .12
12.1 General . 12
12.2 Test report content . 12
Annex A (informative) Checklist for NA sample quality assessment before library construction .13
Annex B (informative) Methods for sample stabilization and storage . 14
Annex C (informative) Bioinformatics pipeline .15
Bibliography . 17

iii
ISO/FDIS 20397-3:2025(en)
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.

iv
ISO/FDIS 20397-3:2025(en)
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 genom
...

ISO/FDIS 20397-3:2025(en)
ISO/TC 276/SC 1/WG 3
Secretariat: ANSI
Date: 2025-02-0304-16
Biotechnology — Massively parallel sequencing — —
Part 3:
General requirements and guidance for metagenomics
FDIS stage
ISO/CDFDIS 20397-3:2023(E2025(en)
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication
may be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying,
or posting on the internet or an intranet, without prior written permission. Permission can be requested from either ISO
at the address below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: + 41 22 749 01 11
Fax: [phone]
EmailE-mail: [e-mail]
Website: www.iso.orgwww.iso.org
Published in Switzerland
ii © ISO 2023 2025 – All rights reserved
ii
ISO/DISFDIS 20397-3:2025(en)
Contents
Foreword . v
Introduction . vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 5
5 Sampling strategy . 6
6 Nucleic acid library preparation . 8
7 Design and review process including sequencing strategy and assessment . 8
8 Database construction . 10
9 Bioinformatics analysis . 11
10 Validation and verification. 12
11 Evaluation . 13
12 Test report . 13
Annex A (informative) Checklist for NA sample quality assessment before library construction15
Annex B (informative) Methods for sample stabilization and storage . 16
Annex C (informative) Bioinformatics pipeline . 17
Bibliography . 21

Foreword . iv
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 5
4.1 General . 5
5 Sampling strategy, including sample handling, transportation, storage and NA processing6
5.1 General . 6
5.2 Primary sample type . 6
5.3 Primary sample stabilization and storage . 7
5.4 Primary sample transportation . 7
5.5 DNA/RNA isolation . 7
5.6 DNA/RNA sample quality . 8
6 Nucleic acid library preparation . 8
iii
ISO/CDFDIS 20397-3:2023(E2025(en)
7 Design and review process including sequencing strategy and assessment . 8
7.1 General . 8
7.2 Short read. 8
7.3 Long read . 8
7.4 Sample preparation and library construction . 9
7.5 Assessment . 9
8 Database construction . 9
8.1 General . 9
8.2 Public database . 9
8.3 Self-build database . 10
9 Bioinformatics analysis . 10
9.1 General . 10
9.2 Identification list of microorganisms . 11
10 Validation and verification. 11
10.1 General . 11
10.2 In silico sequence control for bioinformatics pipeline evaluation . 12
10.3 Real sample control for pipeline evaluation . 12
11 Evaluation . 12
12 Report . 12
12.1 General . 12
12.2 Report content . 13
Annex A (informative) Checklist for NA sample quality assessment before library construction 14
Annex B (informative) Methods for sample stabilization and storage . 15
Annex C (informative) Bioinformatics pipeline . 17
Bibliography . 19
iv © ISO 2023 2025 – All rights reserved
iv
ISO/DISFDIS 20397-3:2025(en)
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.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.htmlwww.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.
Field Code Changed
v
ISO/CDFDIS 20397-3:2023(E2025(en)
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 catalogscatalogues 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.
AnalyzingAnalysing 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.
vi © ISO 2023 2025 – All rights reserved
vi
DRAFT International Stan
...