CEN/TS 17981-1:2023

SLOVENSKI STANDARD
01-marec-2024
In vitro diagnostični delovni postopki Sekvenciranje naslednje generacije (NGS) -
1. del: Preiskava človeškega DNK
In vitro diagnostic Next Generation Sequencing (NGS) workflows - Part 1: Human DNA
examination
Next Generation Sequencing (NGS)-Arbeitsabläufe für die In-vitro-Diagnostik - Teil 1:
Untersuchung von menschlicher DNA
Diagnostic in vitro Séquençage de nouvelle génération (NGS) pour des examens de
l'ADN/ARN humain
Ta slovenski standard je istoveten z: CEN/TS 17981-1:2023
ICS:
11.100.10 Diagnostični preskusni In vitro diagnostic test
sistemi in vitro systems
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

CEN/TS 17981-1
TECHNICAL SPECIFICATION
SPÉCIFICATION TECHNIQUE
November 2023
TECHNISCHE SPEZIFIKATION
ICS 11.100.10
English Version
In vitro diagnostic Next Generation Sequencing (NGS)
workflows - Part 1: Human DNA examination
Diagnostic in vitro Séquençage de nouvelle génération Next Generation Sequencing (NGS)-Arbeitsabläufe für
(NGS) - Partie 1 : Examens de l'ADN humain die In-vitro-Diagnostik - Teil 1: Untersuchung von
menschlicher DNA
This Technical Specification (CEN/TS) was approved by CEN on 15 October 2023 for provisional application.

The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to
submit their comments, particularly on the question whether the CEN/TS can be converted into a European Standard.

CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS
available promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in
parallel to the CEN/TS) until the final decision about the possible conversion of the CEN/TS into an EN is reached.

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, Türkiye and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATIO N

EUROPÄISCHES KOMITEE FÜR NORMUN G

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2023 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 17981-1:2023 E
worldwide for CEN national Members.

Contents Page
European foreword . 4
Introduction . 5
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 7
4 General requirements . 19
4.1 General. 19
4.2 Examination design . 20
4.3 Examination development . 24
4.4 Examination performance verification and validation . 24
4.5 Technical examination performance characteristics . 30
5 Pre-examination processes for examination development . 30
5.1 General. 30
5.2 Human DNA isolation . 32
5.3 DNA sample quality and quantity evaluation . 33
6 Examination processes for examination development . 35
6.1 Sequencing library preparation for examination development . 35
6.2 Sequencing examination development . 40
6.3 Data analysis requirements for examination development . 41
6.4 Quality control (QC) requirements for examination development . 42
7 Requirements for the development of the examination reporting tool . 43
7.1 General. 43
7.2 Report attributes . 44
7.3 Report content . 44
8 Implementation of the in vitro diagnostic NGS workflow into routine practice . 45
9 Reporting and interpretation of results . 46
10 Quality assurance procedures . 47
10.1 General. 47
10.2 Performance monitoring, optimization of the examination and interlaboratory
comparison . 47
Annex A (normative) in vitro diagnostic NGS workflow for single-cell analyses . 48
A.1 General information and requirements on single-cell analyses . 48
A.2 Pre-examination processes for examination development . 49
A.3 Examination phase for examination development. 53
A.4 Implementation of the in vitro diagnostic NGS workflow into routine practice . 54
A.5 Reporting and interpretation of results . 54
A.6 Quality assurance procedures . 54
Annex B (informative) NGS workflow scheme for the examination of DNA . 55
Bibliography . 56

European foreword
This document (CEN/TS 17981-1:2023) has been prepared by Technical Committee CEN/TC 140 “In
vitro diagnostic medical devices”, the secretariat of which is held by DIN.
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.
A list of all parts in this series can be found on the CEN website: www.cencenelec.eu.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
According to the CEN/CENELEC Internal Regulations, the national standards organisations of the
following countries are bound to announce this Technical Specification: 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, Türkiye and the
United Kingdom.
Introduction
Molecular in vitro diagnostics has enabled significant progress in medicine. Further progress is expected
by new technologies analysing profiles of nucleic acids, proteins, and metabolites in human tissues and
body fluids. Next Generation Sequencing (NGS) takes a prominent place in the series of molecular
techniques used for diagnostics. It facilitates sequence analysis of nucleic acids that can result in precise
information for diagnosis and progression of diseases.
The NGS technique, however, has a very complex workflow that contains many steps. The target nucleic
acids can originate from different sources, e.g. tissues, blood, and body fluids. The profiles of the isolated
DNA or methylated DNA can change during specimen collection, transport, storage and processing (e.g.
formalin fixation), making the outcome from diagnostics or research unreliable or even impossible
because the subsequent analytical assay will not determine the situation in the patient but an artificial
profile generated during the pre-examination process. The available material can be small, the cells in a
tissue can be dispersed heterogeneously (e.g. ratio of tumour to normal), the target nucleic acids can be
circulating in blood or body fluids free of cells or in circulating cells (e.g. circulating tumour cells (CTCs)).
For a successful and reliable sequence result, a suitable strategy needs to be chosen for every case
depending on the available material and disease conditions. Therefore, the NGS workflow can differ from
case to case, and the NGS workflow steps need to be carefully considered and chosen to get a sound and
reliable result to determine the best available treatment for the patient. In addition, sequence platforms
can differ in their technique (e.g. detection of a change in a current or fluorescence) and approach (e.g.
panels, short-read sequencing, long-read sequencing) for sequence assessment. The bioinformatics
analysis can differ in approach and ability to detect non-conformities and unreliable sequence results. To
enable such capabilities, NGS metadata needs to be collected during all workflow steps from the patient
to the reporting. In addition, controls and added controls need to be analysed properly. In this way, non-
conformities or detected unreliabilities can be reported to the patient and the treating physician. The
reporting of diagnostic NGS results can differ in clarity and depth, which can lead to different
interpretations.
Standardization of the entire NGS workflow from specimen collection to the reporting of the results to
the patient and the treating physician is needed for the development of reliable NGS examinations.
This document draws upon previous work to standardize the steps for NGS examinations from tissues,
blood and body fluids in what is referred to as the pre-examination phase (sample collection), the
examination phase (library preparation, sequencing), and the post-examination phase (analysis and
reporting).
In this document, the following verbal forms are used:
— “shall” indicates a requirement;
— “should” indicates a recommendation;
— “may” indicates a permission;
— “can” indicates a possibility or a capability.
1 Scope
This document specifies requirements and gives recommendations for next generation sequencing (NGS)
workflows for in vitro diagnostics and biomedical research. This document covers the pre-examination
processes, human DNA (somatic and germline) isolation, sequencing library preparation, sequencing,
sequence analysis and reporting of the examination of sequences for diagnostic purposes from isolated
DNA from, e.g. formalin-fixed and paraffin embedded tissues, fresh frozen tissues, fine needle aspirates
(FNA), whole blood, circulating tumour cells (CTCs), exosomes and other extracellular vesicles,
circulating cell free DNA from plasma, and DNA from saliva.
NOTE 1 Typical applications include, but are not limited to, NGS for oncology, pharmacogenomics and clinical
genetics; approaches include panels (e.g. disease panels, exome panels, target gene panels and in silico panels),
exome and whole genome sequencing, as well as certain epigenetics and certain single-cell analyses.
This document is applicable to molecular in vitro diagnostic examinations including laboratory developed
tests performed by medical laboratories, molecular pathology laboratories and molecular genetic
laboratories. This document is also applicable to laboratory customers, in vitro diagnostics developers
and manufacturers, biobanks, institutions, and organizations performing biomedical research.
This document is not applicable for in situ sequencing, DNA-mediated protein sequencing, forensic
sequencing, sequencing of pathogens or microorganisms and microbiome analysis.
NOTE 2 International, national or regional regulations or requirements or multiples of them can also apply to
specific topics covered in this document.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this documen
...