EN ISO 23418:2022

Microbiology of the food chain - Whole genome sequencing for typing and genomic characterization of bacteria - General requirements and guidance (ISO 23418:2022)

This document specifies the minimum requirements for generating and analysing whole genome sequencing (WGS) data of bacteria obtained from the food chain. This process can include the following stages:
a) handling of bacterial cultures;
b) axenic genomic DNA isolation;
c) library preparation, sequencing, and assessment of raw DNA sequence read quality and storage;
d) bioinformatics analysis for determining genetic relatedness, genetic content and predicting phenotype, and bioinformatics pipeline validation;
e) metadata capture and sequence repository deposition;
f) validation of the end-to-end WGS workflow (fit for purpose for intended application).
This document is applicable to bacteria isolated from:
—    products intended for human consumption;
—    products intended for animal feed;
—    environmental samples from food and feed handling and production areas;
—    samples from the primary production stage.

Mikrobiologie der Lebensmittelkette - Gesamtgenomsequenzierung zur Typisierung und genomischen Charakterisierung von Bakterien - Allgemeine Anforderungen und Leitfaden (ISO 23418:2022)

Dieses Dokument legt die Mindestanforderungen für die Generierung und Analyse von Daten der Gesamtgenomsequenzierung (WGS) für Bakterien, die aus der Lebensmittelkette gewonnen wurden, fest. Dieser Prozess kann die folgenden Phasen umfassen:
a)   Umgang mit Bakterienkulturen;
b)   keimfreie Isolierung der genomischen DNA;
c)   Vorbereitung der genomischen DNA Bibliothek, Sequenzierung und Beurteilung der Read-Qualität von Rohdaten und deren Speicherung;
d)   bioinformatische Analyse zur Bestimmung der genetischen Verwandtschaft, des genetischen Gehalts und zur Vorhersage des Phänotyps sowie Validierung der bioinformatischen Pipeline;
e)   die Erfassung von Metadaten und die Hinterlegung von Sequenzdaten in Repositorien;
f)   Validierung des Gesamt-WGS-Workflows (gebrauchstauglich für die beabsichtigte Anwendung).
Dieses Dokument ist anwendbar auf Bakterien, die isoliert wurden aus:
—   Produkten, die für den menschlichen Verzehr vorgesehen sind;
—   Produkten, die als Futtermittel vorgesehen sind;
—   Umgebungsproben aus Bereichen, in denen Lebensmittel und Futtermittel gehandhabt und hergestellt werden;
—   Proben aus dem Bereich der Primärproduktion.
WARNHINWEIS — Zum Schutz der Gesundheit des Laborpersonals ist es unerlässlich, dass der Umgang mit Bakterienkulturen nur in Laboratorien mit geeigneter Ausstattung und unter der Leitung eines qualifizierten Mikrobiologen erfolgt und dass bei der Entsorgung allen inkubierten Materials mit äußerster Vorsicht vorgegangen wird. Personen, die dieses Dokument anwenden, sollten mit der üblichen Laborpraxis vertraut sein. Dieses Dokument erhebt nicht den Anspruch, alle gegebenenfalls zutreffenden Sicherheitsaspekte im Zusammenhang mit seiner Anwendung zu behandeln. Es liegt in der Verantwortung des Anwenders, angemessene Sicherheits- und Schutzmaßnahmen zu treffen.

Microbiologie de la chaîne alimentaire - Séquençage de génome entier pour le typage et la caractérisation génomique des bactéries - Exigences générales et recommandations (ISO 23418:2022)

Le présent document spécifie les exigences minimales pour générer et analyser des données de séquençage de génome entier (WGS) de bactéries provenant de la chaîne alimentaire. Ce processus peut comprendre les étapes suivantes:
a) manipulation des cultures bactériennes;
b) isolement de l’ADN génomique axène;
c) préparation de la librairie, séquençage et évaluation de la qualité et du stockage des lectures de séquences brutes d’ADN;
d) analyse bioinformatique visant à déterminer la parenté génétique et le contenu génétique, à prédire le phénotype et à valider le pipeline bioinformatique;
e) capture des métadonnées et dépôt dans des bases de données de séquences;
f) validation du processus de WGS de bout en bout (adapté à l’application prévue).
Le présent document est applicable aux bactéries isolées à partir de ce qui suit:
—    des produits destinés à la consommation humaine;
—    des produits destinés à l’alimentation animale;
—    des échantillons environnementaux prélevés dans des zones de production et de manipulation de produits alimentaires et d’aliments pour animaux;
—    des échantillons de production primaire.

Mikrobiologija v prehranski verigi - Sekvenciranje celotnega genoma za tipizacijo in genomsko karakterizacijo bakterij - Splošne zahteve in smernice (ISO 23418:2022)

Ta mednarodni standard določa minimalne zahteve za ustvarjanje in analizo podatkov o sekvenciranju celotnega genoma (WGS), pridobljenih iz bakterij, ki se prenašajo s hrano. Te zahteve se uporabljajo za vse platforme ali kemijske postopke sekvenciranja. Ta proces lahko vključuje naslednje faze:
• ravnanje z bakterijskimi kulturami;
• izolacija genomske DNA;
• oblikovanje knjižnice sekvenciranja, sekvenciranje in ocena kakovosti odčitavanja in shranjevanja neobdelanih sekvenc DNK;
• bioinformacijska analiza, vključno z metodami, kot so visokokakovostna analiza polimorfizma posameznega nukleotida (hqSNP), tipizacija na osnovi zaporedij lokusov osrednjega in celotnega genoma (cgMLST, wgMLST) ter validacija bioinformacijskih algoritemskih procesov za obdelavo neobdelanih podatkov o sekvenciranju; in
• zajemanje metapodatkov in shranjevanje sekvenc v repozitorij.

General Information

• Status: Published
• Publication Date: 21-Jun-2022
• Withdrawal Date: 30-Dec-2022
• ICS: 07.100.30 - Food microbiology
• Technical Committee: CEN/TC 463 - Microbiology of the food chain
• Drafting Committee: CEN/TC 463 - Microbiology of the food chain
• Current Stage: 6060 - Definitive text made available (DAV) - Publishing
• Start Date: 22-Jun-2022
• Completion Date: 22-Jun-2022
• Directive: 882/2004 - Regulation (EC) No 882/2004 of the European Parliament and of the Council of 29 April 2004 onofficial controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules
• Ref Project: SIST EN ISO 23418:2022 - Microbiology of the food chain - Whole genome sequencing for typing and genomic characterization of bacteria - General requirements and guidance (ISO 23418:2022)

Standards Content (Sample)

SLOVENSKI STANDARD
SIST EN ISO 23418:2022
01-september-2022

Mikrobiologija v prehranski verigi - Sekvenciranje celotnega genoma za tipizacijo

in genomsko karakterizacijo bakterij - Splošne zahteve in smernice (ISO
23418:2022)
Microbiology of the food chain - Whole genome sequencing for typing and genomic

characterization of bacteria - General requirements and guidance (ISO 23418:2022)

Mikrobiologie der Lebensmittelkette - Gesamtgenomsequenzierung zur Typisierung und

genomischen Charakterisierung von Bakterien - Allgemeine Anforderungen und
Leitfaden (ISO 23418:2022)

Microbiologie de la chaîne alimentaire - Séquençage de génome entier pour le typage et

la caractérisation génomique des bactéries - Exigences générales et recommandations

(ISO 23418:2022)
Ta slovenski standard je istoveten z: EN ISO 23418:2022
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST EN ISO 23418:2022 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 23418:2022
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SIST EN ISO 23418:2022
EN ISO 23418
EUROPEAN STANDARD
NORME EUROPÉENNE
June 2022
EUROPÄISCHE NORM
ICS 07.100.30
English Version
Microbiology of the food chain - Whole genome
sequencing for typing and genomic characterization of
bacteria - General requirements and guidance (ISO
23418:2022)

Microbiologie de la chaîne alimentaire - Séquençage de Mikrobiologie der Lebensmittelkette - Vollständige

génome entier pour le typage et la caractérisation Genomsequenzierung zur Typisierung und

génomique des bactéries - Exigences générales et genomischen Charakterisierung von Bakterien in

recommandations (ISO 23418:2022) Lebensmitteln - Allgemeine Anforderungen und
Leitfaden (ISO 23418:2022)
This European Standard was approved by CEN on 20 May 2022.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this

European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references

concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN

member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by

translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management

Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,

Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and

United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2022 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 23418:2022 E

worldwide for CEN national Members.
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SIST EN ISO 23418:2022
EN ISO 23418:2022 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

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SIST EN ISO 23418:2022
EN ISO 23418:2022 (E)
European foreword

This document (EN ISO 23418:2022) has been prepared by Technical Committee ISO/TC 34 "Food

products" in collaboration with Technical Committee CEN/TC 463 “Microbiology of the food chain” the

secretariat of which is held by AFNOR.

This European Standard shall be given the status of a national standard, either by publication of an

identical text or by endorsement, at the latest by December 2022, and conflicting national standards

shall be withdrawn at the latest by December 2022.

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. CEN shall not be held responsible for identifying any or all such patent rights.

This document has been prepared under a Standardization Request given to CEN by the European

Commission and the European Free Trade Association.

Any feedback and questions on this document should be directed to the users’ national standards

body/national committee. A complete listing of these bodies can be found on the CEN website.

According to the CEN-CENELEC Internal Regulations, the national standards organizations of the

following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,

Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,

Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of

North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the

United Kingdom.
Endorsement notice

The text of ISO 23418:2022 has been approved by CEN as EN ISO 23418:2022 without any modification.

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SIST EN ISO 23418:2022
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SIST EN ISO 23418:2022
INTERNATIONAL ISO
STANDARD 23418
First edition
2022-06
Microbiology of the food chain —
Whole genome sequencing for typing
and genomic characterization of
bacteria — General requirements and
guidance
Microbiologie de la chaîne alimentaire — Séquençage de génome
entier pour le typage et la caractérisation génomique des bactéries —
Exigences générales et recommandations
Reference number
ISO 23418:2022(E)
© ISO 2022
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SIST EN ISO 23418:2022
ISO 23418:2022(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2022

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
© ISO 2022 – All rights reserved
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SIST EN ISO 23418:2022
ISO 23418:2022(E)
Contents Page

Foreword ..........................................................................................................................................................................................................................................v

Introduction .............................................................................................................................................................................................................................. vi

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ..................................................................................................................................................................................... 1

3 Terms and definitions .................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 6

4.1 General ........................................................................................................................................................................................................... 6

4.2 Laboratory operation: sample preparation and sequencing ......................................................................... 6

4.3 Bioinformatics analysis .................................................................................................................................................................. 7

4.3.1 General ........................................................................................................................................................................................ 7

4.3.2 SNP analyses .......................................................................................................................................................................... 7

4.3.3 MLST analyses ...................................................................................................................................................................... 7

4.3.4 Kmer distance analysis ................................................................................................................................................ 7

4.4 Metadata formats and sequence repository deposition .................................................................................... 7

4.5 Validation and verification of WGS workflow ............................................................................................................. 8

5 General laboratory guidance .................................................................................................................................................................. 8

5.1 Bacterial isolation and DNA extraction ............................................................................................................................ 8

5.2 Laboratory environment ............................................................................................................................................................... 8

5.3 Standard operating procedures and nonconforming work ............................................................................ 8

5.4 Laboratory information management system ........................................................................................................... 8

5.5 Laboratory competence.................................................................................................................................................................. 8

6 Laboratory operations ........................................................................................................................................... ........................................9

6.1 Sample preparation and storage ............................................................................................................................................ 9

6.2 Bacterial isolates .................................................................................................................................................................................. 9

6.3 DNA isolation ........................................................................................................................................................................................... 9

6.4 Library preparation and sequencing .................................................................................................................................. 9

6.4.1 Library preparation ......................................................................................................................................................... 9

6.4.2 DNA sequencing ............................................................................................................................................................... 10

6.4.3 Use of controls ................................................................................................................................................................... 10

6.4.4 Assessing raw read data quality ....................................................................................................................... 10

6.4.5 Sample and data storage and retention ...................................................................................................... 10

7 Bioinformatic data analysis ..................................................................................................................................................................11

7.1 Requirements for software and bioinformatic pipelines used for data analysis ..................... 11

7.2 Logging and documentation .................................................................................................................................................... 11

7.3 Quality assessments ....................................................................................................................................................................... 11

7.4 SNP analyses ..........................................................................................................................................................................................12

7.5 MLST analyses (cgMLST and wgMLST) ........................................................................................................................12

7.6 Target gene detection ....................................................................................................................................................................13

7.7 Phylogenetic tree or dendrogram generation.......................................................................................................... 13

7.8 Metrics and log files ........................................................................................................................................................................13

7.9 Interpreting and reporting the results of bioinformatics analyses .....................................................13

7.9.1 Interpreting results from bioinformatics pipelines .........................................................................13

7.9.2 Reporting genomic analysis results ............................................................................................................... 14

8 Metadata ....................................................................................................................................................................................................................14

8.1 General ........................................................................................................................................................................................................ 14

8.2 Metadata interoperability and future-proofing .................................................................................................... 14

8.2.1 General ..................................................................................................................................................................................... 14

8.2.2 Ontologies .............................................................................................................................................................................. 14

8.2.3 ISO WGS Slim ...................................................................................................................................................................... 14

8.3 Formatting metadata using this document ............................................................................................................... 15

8.4 Metadata associated with sample collection ............................................................................................................ 15

iii
© ISO 2022 – All rights reserved
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SIST EN ISO 23418:2022
ISO 23418:2022(E)

8.5 Metadata associated with the isolate .............................................................................................................................. 16

8.6 Metadata associated with the sequence ....................................................................................................................... 17

9 Sequence repositories .................................................................................................................................................................................19

10 Validation and verification ....................................................................................................................................................................20

10.1 Validation ................................................................................................................................................................................................. 20

10.1.1 General .....................................................................................................................................................................................20

10.1.2 Validation of laboratory operations ............................................................................................................... 21

10.1.3 Validation of the bioinformatics pipeline .................................................................................................. 21

10.1.4 Validation of the end-to-end workflow ....................................................................................................... 22

10.2 Verification ...................................................................... ........................................................................................................................ 22

10.2.1 General .....................................................................................................................................................................................22

10.2.2 Verification of laboratory operations ........................................................................................................... 22

10.2.3 Verification of the bioinformatics pipeline ..............................................................................................22

Annex A (informative) Development of quality metrics and use of controls .........................................................24

Annex B (informative) Laboratory contact information fields ............................................................................................29

Annex C (informative) Geographic location of sample collection fields .....................................................................31

Annex D (informative) Isolate passage history fields ....................................................................................................................32

Annex E (informative) Antibiogram results and methods fields .......................................................................................33

Annex F (informative) Virulence factor detection and methods fields .......................................................................35

Annex G (informative) Sequence quality control metrics ..........................................................................................................36

Annex H (informative) Metadata specification .....................................................................................................................................37

Annex I (informative) Instructions for ontology slim integration by software developers ..................40

Bibliography ............................................................................................................................................................................................................................ 44

© ISO 2022 – All rights reserved
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SIST EN ISO 23418:2022
ISO 23418:2022(E)
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 documents 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).

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www.iso.org/patents).

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 34, Food products, Subcommittee SC 9,

Microbiology, in collaboration with the European Committee for Standardization (CEN) Technical

Committee CEN/TC 463, Microbiology of the food chain, in accordance with the Agreement on technical

cooperation between ISO and CEN (Vienna Agreement).

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.
© ISO 2022 – All rights reserved
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SIST EN ISO 23418:2022
ISO 23418:2022(E)
Introduction

Next generation sequencing (NGS) provides rapid, economical and high-throughput access to

microbial whole genome sequences and is being applied to an expanding number of problems in food

microbiology. Whole genome sequences are representations of the biological potential of the sequenced

organism at single base resolution. Whole genome sequencing (WGS) offers significant advantages over

existing technologies (e.g. serotyping, pulsed field gel electrophoresis, antibiotic resistance phenotype)

for many applications. WGS-based analyses are used by public health laboratories to detect outbreaks,

and to detect mutations, genes and other genetic features to characterize virulence and survival

potential. Within the food industry, there is interest in using whole genome sequences to characterize

bacterial isolates from ingredients and environmental surfaces, to better understand their origin and

ecology, and to update procedures to reduce risk. Some companies have developed, or are developing,

the capacity to collect and analyse whole genome sequence data. Others are turning to third-party

laboratories to perform these services, as they have done for other microbiological analyses.

This document provides guidance for both the laboratory and bioinformatic components of whole

genome sequences and associated metadata for bacterial foodborne microorganisms sampled along

the food chain (e.g. ingredients, food, feed, production environment). Although microbiology of the

food chain includes viruses and fungi, this document is only intended for bacteria. This document is

intended to be applicable to all currently available next generation DNA sequencing technologies. It

may be applied to analysis of whole genome sequence data with proprietary, open-source or custom

software. It is not intended to specify sequencing chemistries, analytical methods or software. This

document defines laboratory, data and metadata stewardship practices to ensure that analyses are

clearly reported, transparent and open to inquiry. This document is for use by laboratories to develop

their management systems for quality and technical operations. Laboratory customers and regulatory

authorities can also use it in confirming or recognizing the competence of laboratories. This document

can also be applied in other domains (e.g. environment, human health, animal health).

© ISO 2022 – All rights reserved
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SIST EN ISO 23418:2022
INTERNATIONAL STANDARD ISO 23418:2022(E)
Microbiology of the food chain — Whole genome
sequencing for typing and genomic characterization of
bacteria — General requirements and guidance

WARNING — In order to safeguard the health of laboratory personnel, it is essential that

handling of bacterial cultures is only undertaken in properly equipped laboratories, under the

control of a skilled microbiologist, and that great care is taken in the disposal of all incubated

materials. Persons using this document should be familiar with normal laboratory practice.

This document does not purport to address all safety aspects, if any, associated with its use. It is

the responsibility of the user to establish appropriate safety and health practices.

1 Scope

This document specifies the minimum requirements for generating and analysing whole genome

sequencing (WGS) data of bacteria obtained from the food chain. This process can include the following

stages:
a) handling of bacterial cultures;
b) axenic genomic DNA isolation;

c) library preparation, sequencing, and assessment of raw DNA sequence read quality and storage;

d) bioinformatics analysis for determining genetic relatedness, genetic content and predicting

phenotype, and bioinformatics pipeline validation;
e) metadata capture and sequence repository deposition;

f) validation of the end-to-end WGS workflow (fit for purpose for intended application).

This document is applicable to bacteria isolated from:
— products intended for human consumption;
— products intended for animal feed;
— environmental samples from food and feed handling and production areas;
— samples from the primary production stage.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminology databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
© ISO 2022 – All rights reserved
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SIST EN ISO 23418:2022
ISO 23418:2022(E)
3.1
adapter sequence

DNA with a known sequence that is added to the end of a DNA library fragment to facilitate the

sequencing process (e.g. annealing to a flow cell)
3.2
annotation
process of identifying genes and other features on genome assemblies (3.4)
3.3
antibiogram

summary of antimicrobial susceptibility testing results performed for a specific microorganism,

usually represented in tabular form
3.4
assembly

output from process of aligning and merging sequencing reads (3.38) into larger contiguous sequences

(contigs (3.10))
3.5
base calling

process of assigning nucleotides and quality scores to positions in sequencing reads (3.38)

3.6
bioinformatics
collection, storage and analysis of biological data including sequences
3.7
bioinformatics pipeline

individual programs, scripts or pieces of software linked together, where output from one program is

used as input for the next step in data processing
3.8
carryover-contamination

sample contamination linked to previous experiments, transferred to the current analysis (e.g.

carryover-contamination from amplification products in prior polymerase chain reaction (PCR)

experiments to the current PCR analysis, or carryover-contamination of previously sequenced samples

from one sequencing run to another)
3.9
Chemical Entities of Biological Interest Ontology
ChEBI
ontology (3.35) for describing small chemical compounds
3.10
contig

contiguous stretch of DNA sequence that results from the assembly (3.4) of smaller, overlapping DNA

sequence reads (3.38)
3.11
controlled vocabulary
finite set of values that represent the only allowed values for a data item
[SOURCE: ISO 11238:2018, 3.18, modified — Note 1 to entry deleted.]
3.12
coverage
number of times that a given base position is read in a sequencing run
Note 1 to entry: The number of reads (3.38) that cover a particular position.
© ISO 2022 – All rights reserved
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SIST EN ISO 23418:2022
ISO 23418:2022(E)

[SOURCE: ISO 20397-2:2021, 3.6, modified — Admitted term “coverage depth” deleted.]

3.13
cross-contamination

contamination of a sample (bacterial isolate (3.23) or DNA) with other samples during the preparation

of a sequencing run
3.14
DNA sample
portio
...

SLOVENSKI STANDARD
oSIST prEN ISO 23418:2020
01-november-2020

Mikrobiologija v prehranski verigi - Sekvenciranje celotnega genoma za tipizacijo

in genomsko karakterizacijo bakterij v živilih - Splošne zahteve in navodilo
(ISO/DIS 23418:2020)
Microbiology of the food chain - Whole genome sequencing for typing and genomic

characterization of foodborne bacteria - General requirements and guidance (ISO/DIS

23418:2020)

Mikrobiologie der Lebensmittelkette - Vollständige Genomsequenzierung zur Typisierung

und genomischen Charakterisierung von Bakterien in Lebensmitteln - Allgemeine
Anforderungen und Leitfaden (ISO/DIS 23418:2020)

Microbiologie de la chaîne alimentaire - Séquençage de génome complet pour le typage

et la caractérisation génomique des bactéries dans les aliments - Exigences générales

et recommandations (ISO/DIS 23418:2020)
Ta slovenski standard je istoveten z: prEN ISO 23418
ICS:
07.100.30 Mikrobiologija živil Food microbiology
oSIST prEN ISO 23418:2020 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN ISO 23418:2020
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oSIST prEN ISO 23418:2020
DRAFT INTERNATIONAL STANDARD
ISO/DIS 23418
ISO/TC 34/SC 9 Secretariat: AFNOR
Voting begins on: Voting terminates on:
2020-09-18 2020-12-11
Microbiology of the food chain — Whole genome
sequencing for typing and genomic characterization of
foodborne bacteria — General requirements and guidance

Microbiologie de la chaîne alimentaire — Séquençage de génome complet pour le typage et la

caractérisation génomique des bactéries dans les aliments — Exigences générales et recommandations

ICS: 07.100.30
THIS DOCUMENT IS A DRAFT CIRCULATED
This document is circulated as received from the committee secretariat.
FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS
ISO/CEN PARALLEL PROCESSING
BEING ACCEPTABLE FOR INDUSTRIAL,
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NATIONAL REGULATIONS.
ISO/DIS 23418:2020(E)
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NOTIFICATION OF ANY RELEVANT PATENT
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PROVIDE SUPPORTING DOCUMENTATION. ISO 2020
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© ISO 2020

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Contents Page

Foreword ..........................................................................................................................................................................................................................................v

Introduction ................................................................................................................................................................................................................................vi

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 6

4.1 General ........................................................................................................................................................................................................... 6

4.2 Laboratory operation: sample preparation and sequencing .......................................................................... 6

4.3 Bioinformatics analysis ................................................................................................................................................................... 6

4.4 Metadata formats and sequence repository deposition...................................................................................... 7

4.5 Validation and verification of WGS Workflow .............................................................................................................. 7

5 General laboratory guidance ................................................................................................................................................................... 7

5.1 Bacterial isolation and DNA extraction .............................................................................................................................. 7

5.2 Laboratory environment ................................................................................................................................................................ 7

5.3 Standard Operating Procedures (SOPs) and non-conforming work ........................................................ 7

5.4 Laboratory information management system (LIMS) .......................................................................................... 8

5.5 Laboratory competence .................................................................................................................................................................. 8

6 Laboratory operations ................................................................................................................................................................................... 8

6.1 Sample preparation and storage ............................................................................................................................................. 8

6.2 Bacterial isolates ................................................................................................................................................................................... 8

6.3 DNA Isolation ........................................................................................................................................................................................... 8

6.4 Library preparation ............................................................................................................................................................................ 9

6.4.1 DNA sequencing ............................................................................................................................................................... 9

6.4.2 Use of controls ................................................................................................................................................................... 9

6.4.3 Assessing raw read data quality ......................................................................................................................... 9

6.4.4 Sample and data storage and retention ....................................................................................................10

7 Bioinformatic data analysis ...................................................................................................................................................................10

7.1 Requirements for software and/or bioinformatic pipelines used for data analysis ................10

7.2 Logging and documentation .....................................................................................................................................................10

7.3 Quality assessments ........................................................................................................................................................................10

7.4 SNP analyses ..........................................................................................................................................................................................11

7.5 MLST analyses (cgMLST and wgMLST) ..........................................................................................................................12

7.6 Target gene detection .....................................................................................................................................................................12

7.7 Phylogenetic tree or dendrogram generation ...........................................................................................................12

7.8 Metrics and log files .........................................................................................................................................................................12

7.9 Interpreting and reporting the results of bioinformatics analyses ........................................................13

7.9.1 Interpreting results from bioinformatics pipelines ........................................................................13

7.9.2 Reporting genomic analysis results ..............................................................................................................13

8 Metadata ....................................................................................................................................................................................................................13

8.1 General ........................................................................................................................................................................................................13

8.2 Metadata Interoperability and Future-Proofing .....................................................................................................13

8.2.1 Ontologies ...........................................................................................................................................................................14

8.2.2 ISO WGS Slim ....................................................................................................................................................................14

8.3 Formatting Metadata Using the Standard.....................................................................................................................14

8.4 Metadata associated with sample collection .............................................................................................................14

8.5 Metadata associated with the isolate ...............................................................................................................................14

8.6 Metadata associated with the sequence ........................................................................................................................14

9 Sequence repositories .................................................................................................................................................................................14

10 Validation and verification .....................................................................................................................................................................15

10.1 Validation ..................................................................................................................................................................................................15

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10.1.1 Validation of laboratory operations .............................................................................................................15

10.1.2 Validation of the bioinformatics pipeline ................................................................................................15

10.1.3 Validation of the end-to-end workflow .....................................................................................................16

10.2 V erification ..............................................................................................................................................................................................16

10.2.1 Verification of laboratory operations ..........................................................................................................16

10.2.2 Verification of the bioinformatics pipeline.............................................................................................17

Annex A (informative) .....................................................................................................................................................................................................23

Annex B (informative) Laboratory Contact Information Fields ............................................................................................27

Annex C (informative) Geographic Location of Sample Collection Fields ...................................................................29

Annex D (informative) Isolate passage history fields ......................................................................................................................30

Annex E (informative) Antibiogram Results and Methods Fields........................................................................................31

Annex F (informative) Virulence Factor Detection and Methods Fields .......................................................................33

Annex G (informative) Sequence Quality Control Metrics...........................................................................................................34

Annex H (informative) Metadata specification .......................................................................................................................................35

Annex I (informative) Instructions for Ontology Slim Integration by Software Developers ...................38

Bibliography .............................................................................................................................................................................................................................42

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Foreword

ISO (the International Organizations 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 documents 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).

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www .iso .org/ patents).

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 34, Food Products, Subcommittee SC 9,

Microbiology.

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.
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Introduction

Next generation sequencing (NGS) provides rapid, economical and high-throughput access to microbial

whole genome sequences (WGS) and is being applied to an expanding number of problems in food

microbiology. WGS are digital representations of the biological potential of the sequenced organism

at single base resolution. The digital nature of WGS data is a departure from the continuous nature of

phenotypes routinely analyzed in food microbiology. Therefore, WGS offers significant advantages over

existing technologies (e.g., serology, pulsed field gel electrophoresis, antibiotic resistance phenotype).

WGS-based analyses are used by public health laboratories to detect outbreaks, and to detect mutations,

genes and other genetic features to characterize virulence and survival potential. Within the food

industry, there is interest in WGS to characterize bacterial isolates from outsourced ingredients and

environmental surfaces, to better understand their origin and ecology, and to update procedures to

reduce risk. Some companies have developed, or are developing, the capacity to collect and analyze

WGS data. Others will turn to third party laboratories to perform these services, as they currently do

for other microbiological analyses.
Removed text

This standard is intended to provide guidance for both the laboratory and bioinformatic components

of WGS and associated metadata for foodborne microorganisms. This standard is intended to be

applicable to all currently available short- and long-read DNA sequencing technologies. It may be

applied to analysis of WGS data with proprietary, open-source, and custom software. It is not intended

to specify sequencing chemistries, analytical methods, or software. The standard defines laboratory,

data, and metadata stewardship practices to ensure that analyses are clearly reported, transparent,

open to inquiry, and available for unanticipated uses. This standard is for use by laboratories to develop

their management systems for quality and technical operations. Laboratory customers and regulatory

authorities may also use it in confirmation or recognizing the competence of laboratories.

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DRAFT INTERNATIONAL STANDARD ISO/DIS 23418:2020(E)
Microbiology of the food chain — Whole genome
sequencing for typing and genomic characterization of
foodborne bacteria — General requirements and guidance
1 Scope

This international standard specifies minimum requirements for generating and analyzing whole-

genome sequencing (WGS) data obtained from foodborne bacteria. These requirements are applicable

to any sequencing platform or chemistry. This process may include the following stages:

a) Handling of bacterial cultures;
b) Genomic DNA isolation;

c) Library preparation, sequencing, and assessment of raw DNA sequence read quality and storage;

d) Bioinformatics analysis for determining genetic relatedness, genetic content and predicting

phenotype, and bioinformatics pipeline validation;
e) Metadata capture and sequence repository deposition; and

f) Validation of the end-to-end WGS workflow (fit for purpose for intended application).

2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
adapter sequence

DNA with a known sequence, which is added to the end of a DNA library fragment, to facilitate the

sequencing process (e.g., annealing to a flow cell)
3.2
annotation
process of identifying genes and other features on genome assemblies
3.3
antibiogram

summary of antimicrobial susceptibility testing results performed for a specific microorganism,

usually represented in tabular form
3.4
assembly

output from process of aligning and merging sequencing reads into larger contiguous sequences

(contigs)
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3.5
base calling

process of assigning nucleotides and quality scores to positions in sequencing reads

3.6
bioinformatics
collection, storage, and analysis of biological sequence data
3.7
bioinformatics pipeline

individual programs, scripts, or pieces of software linked together, where output from one program is

used as input for the next step in data processing
3.8
carryover-contamination

samples contaminated with DNA from previously sequenced samples, or substances, including EDTA,

phenol-chloroform, protein, excess salts
3.9
Chemical Entities of Biological Interest Ontology
ChEBI
ontology for describing small chemical compounds
3.10
contig

contiguous stretch of DNA sequence that results from the assembly of smaller, overlapping DNA

sequence reads
3.11
controlled vocabulary
finite set of values that represent the only allowed values for a data item
[SOURCE: ISO 11238:2018(en)]
3.12
coverage
average number of times each base in a genome is sequenced
3.13
cross-contamination
contamination of a sample (bacterial isolate or DNA) with other samples
3.14
DNA quality

indication of DNA purity (free of polysaccharides, contaminants and enzyme inhibitors) and integrity

(high molecular weight with little to no evidence of degradation)
3.15
DNA Sample
portion of DNA extracted from some material
3.16
draft assembly

de novo genome assembly consisting of contigs with no implied order, typically generated using whole-

genome shotgun sequencing with a short-read technology
3.17
Environment Ontology
EnvO
ontology for describing environmental features and habitats
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3.18
FoodEx2 Ontology
FoodEx2

standardised food classification and description system developed by the European Food Safety

Authority (EFSA)
3.19
Food Ontology
FoodOn
ontology for describing food products, animal feed and food processing
3.20
Gazetteer Ontology
GAZ
ontology for describing geographical locations
3.21
index

oligonucleotide sequences used in the process of library preparation to tag or barcode DNA from

specific samples, so that multiple samples may be combined (multiplexed) in a sequencing reaction

3.22
International Nucleotide Sequence Database Collaboration
INSDC

initiative operated by the DNA Database of Japan (DDBJ), the European Molecular Biology Laboratory,

European Bioinformatics Institute (EMBL-EBI) and the National Center for Biotechnology

Information (NCBI)
3.23
ISO WGS Slim

ontology Slim containing interoperable fields and terms pertaining to the use of WGS for food

microbiology
3.24
isolate
population of bacterial cells in pure culture derived from a single colony
3.25
kmers
all possible sequences of length k that are contained in a whole genome sequence
3.26
library

collection of genomic DNA fragments from a single isolate intended for determining genome sequence

3.27
management system

quality, administrative and technical systems that govern the operations of an organization

Note 1 to entry: For the purposes of this document organization refers to the laboratory

3.28
mapping
use of software to align sequencing reads to reference sequences
3.29
metadata
data that describes and defines other data
[SOURCE: ISO/IEC 11179-1:2015, 3.2.16]
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3.30
minimal data for matching
MDM

information required to describe the sample source and provenance of a genomic sequence, as defined

[10]

by the Global Microbial Identifier , and implemented by the International Nucleotide Sequence

Database Collaboration
3.31
minimum inhibitory concentration
MIC

lowest concentration that, under defined in vitro test conditions, reduces growth by an agreed amount

within a defined period of time.
Note 1 to entry: to entry The MIC is expressed in mg/l.
[SOURCE: ISO 16256:2012(en)]
3.32
multi-locus sequence typing
MLST

method of genomic analysis in which nucleotide variants within predefined sets of loci, either core

genome loci for cgMLST or whole genome loci for wgMLST, are identified
3.33
N50

length (N) such that sequence contigs of N or longer include half the bases in the assembly

3.34
NCBITaxon Ontology
NCBITaxon
automatic translation of the NCBI taxonomy database
3.35
NG50

length (N) of DNA such that sequence contigs of N or longer include half the bases in the genome

3.36
Open Biological and Biomedical Ontology Foundry
OBO Foundry

collection of ontologies created by a collective of ontology developers that are committed to

collaboration and adherence to shared principles
3.37
ontology

controlled vocabulary arranged in a hierarchy, where the terms are connected by logical relationships

3.38
ontology Slim

set of ontology fields and terms annotated as part of a particular collection, often for a specific purpose,

which can be extracted to create a file distinct from the original ontology
3.39
Phred (Q) sequence quality score

measure of the probability that a base is incorrectly assigned at a given position in the sequence

expressed as:
QP=−10log

Note 1 to entry: to entry A score of Q30 indicates that there is a 1 in 1000 chance that a base is incorrectly

assigned (i.e. the base call is 99.9 % accurate)
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3.40
read
Nucleotide sequence inferred from a fragment of DNA or RNA
3.41
sequence repository
database in which WGS datasets are stored and managed

Note 1 to entry: to entry A public repository allows unrestricted access to the data, while a private or federated

repository restricts access to the data
3.42
sequencing replicate, biological

sequencing a different colony from the same isolate obtained from the same sample material, to assess

biological variation
3.43
sequencing replicate, technical

resequencing of the same biological sample or library to assess sequence variation due to

instrumentation and protocol
3.44
serotype

classification scheme based on the antigenic detection or sequence-based detection of genes encoding

bacteria surface molecules
3.45
Single Nucleotide Polymorphism
SNP
a SNV that passes a particular quality and/or frequency threshold
3.46
Single Nucleotide Variant
SNV

differences between the nucleotide states at the same genomic position of two or more isolates

3.47
strain

the descendants of a single isolation in pure culture, usually derived from a single initial colony on a

[1]
solid growth medium

Note 1 to entry: to entry A strain may be considered an isolate or group of isolates that can be distinguished

from other isolates of the same genus and species by phenotypic and genotypic characteristics

3.48
validation

establishment of the performance characteristics of a method and provision of objective evidence that

the performance requirements for a specified intended use are fulfilled
[SOURCE: ISO 16140-1:2016(en)]
3.49
validated data entry
automated process ensuring that data entered into a repository is correct
3.50
verification

demonstration that a validated method functions in the user's hands according to the method's

specifications determined in the validation (3.48) study and is fit for its purpose

[SOURCE: ISO 16140-1:2016(en)]
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3.51
whole genome sequencing
WGS

process of determining the DNA sequence of an organism’s genome using total genomic DNA as input

4 Principle
4.1 General
Any organization that handles samples, performs s
...