ISO 20397-1:2022

Biotechnology - Massively parallel sequencing - Part 1: Nucleic acid and library preparation

This document specifies the general requirements for and gives guidance on quality assessments of nucleic acid samples. It specifies general guidelines for library preparations and library quality assessments prior to sequencing and data generation.

Biotechnologie — Séquençage parallèle massif — Partie 1: Acides nucléiques et préparation des collections

General Information

• Status: Published
• Publication Date: 16-Mar-2022
• ICS: 07.080 - Biology. Botany. Zoology
• Technical Committee: ISO/TC 276 - Biotechnology
• Drafting Committee: ISO/TC 276/WG 3 - Analytical methods
• Current Stage: 6060 - International Standard published
• Start Date: 17-Mar-2022
• Due Date: 11-Jun-2023
• Completion Date: 17-Mar-2022

Relations

• Consolidated By: ISO 13164-4:2023 - Water quality - Radon-222 - Part 4: Test method using two-phase liquid scintillation counting
• Effective Date: 06-Jun-2022

Overview

ISO 20397-1:2022 - Biotechnology - Massively parallel sequencing - Part 1: Nucleic acid and library preparation - specifies general requirements and provides guidance for evaluating nucleic acid sample quality and for preparing sequencing libraries prior to data generation. The standard focuses on upstream steps that directly affect massively parallel sequencing (MPS) results, including nucleic acid quantification, purity, integrity, library construction, and quality assessment.

Key Topics and Requirements

• Nucleic acid sample quality evaluation
  • Establish and document workflows for accurate, reproducible quantification.
  • Assess sample quantity, purity and integrity using validated methods.
  • Recommended analytical approaches include gel electrophoresis, capillary electrophoresis, microfluidic systems and PCR-based checks (as guidance, not prescriptive thresholds).
• Library preparation
  • General guidelines for fragmentation (mechanical, enzymatic, chemical), size distribution and purification.
  • Addition of universal sequences via end repair and adapter ligation, and strategies for barcoding/indexing multiple samples.
  • Size selection, amplification, purification/cleanup steps and library qualification.
• Library and sample quantification
  • Guidance on selecting appropriate quantification methods and developing QC procedures to ensure libraries are suitable for sequencing.
• Validation and controls
  • Use of positive, negative, no-template and spike-in controls; reference materials to monitor process performance.
• Contamination prevention
  • Requirements for primary sample evaluation, protocol design and operational procedures to minimize contamination risk.
• Supporting material
  • Informative annexes include a checklist for sample quality assessment, examples of platform-specific quality criteria, and a reference material list.

Applications and Who Uses It

ISO 20397-1:2022 is applicable to organizations performing MPS workflows where upstream sample handling and library prep influence downstream data quality:

• Molecular biology and genomics laboratories
• Clinical and diagnostic sequencing facilities
• Contract research organizations (CROs) and biotech companies
• Public health and research institutions performing high-throughput sequencing
• Laboratory managers and QA/QC personnel developing validated sequencing workflows

Adopting the standard helps improve reproducibility of sequencing data, reduce failed runs and contamination events, and supports compliance with laboratory quality systems.

Related Standards

• ISO 20395:2019 - Requirements for evaluating performance of nucleic acid quantification methods (qPCR/dPCR) - referenced for quantification performance.
• Other parts of the ISO 20397 series (see ISO website) address additional MPS topics and platform-specific considerations.

Keywords: ISO 20397-1:2022, massively parallel sequencing, MPS, nucleic acid quality, library preparation, library quality assessment, sequencing workflows, adapter ligation, barcoding/indexing, library quantification.

Standards Content (Sample)

INTERNATIONAL ISO
STANDARD 20397-1
First edition
2022-03
Biotechnology — Massively parallel
sequencing —
Part 1:
Nucleic acid and library preparation
Biotechnologie — Séquençage parallèle massif —
Partie 1: Acides nucléiques et préparation des collections
Reference number
© 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
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or ISO’s member body in the country of the requester.
ISO copyright office
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Published in Switzerland
ii
Contents Page
Foreword .v
Introduction . vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Nucleic acid sample quality evaluation . 2
4.1 General . 2
4.2 Sample quantification . 3
4.3 Sample purity . 3
4.4 Sample integrity . 3
4.4.1 General . 3
4.4.2 Agarose gel electrophoresis . 3
4.4.3 Capillary gel electrophoresis . 3
4.4.4 Microfluidic analysis system . 3
4.4.5 PCR method . . 3
5 Nucleic acid library preparation .4
5.1 General . 4
5.2 Fragmentation . 4
5.2.1 General . 4
5.2.2 Mechanical fragmentation . 4
5.2.3 Enzymatic fragmentation . 4
5.2.4 Chemical fragmentation . 5
5.2.5 Fragmented nucleic acid sample quantity . 5
5.2.6 Fragmented nucleic acid sample purity . 5
5.2.7 Fragmented nucleic acid size distribution . 5
5.2.8 Fragmented nucleic acid purification using gel electrophoresis. 5
5.3 Addition of universal sequences . 5
5.3.1 Repair . 5
5.3.2 Ligation of adapter . 5
5.3.3 Barcoding/indexing. 6
5.4 Size selection . 6
5.5 Amplification . 6
5.6 Purification and clean up procedures . 6
5.7 Library quantification . . 7
5.7.1 Library quantification method . 7
5.7.2 Selection of quantification method . 7
5.8 Library qualification . 7
5.8.1 General . 7
5.8.2 Methods . 7
6 Validation . . 7
7 Reference materials or controls . 8
7.1 General . 8
7.2 Control samples . 8
7.3 Positive control . 8
7.4 Negative control . 9
7.5 No-template control . 9
7.6 Spike-in control . 9
7.7 Reference materials . 9
8 Contaminations .9
8.1 General . 9
8.2 Primary sample evaluation . 9
iii
8.3 Protocol and operation procedure. 10
Annex A (informative) Checklist for sample quality assessment before library construction .11
Annex B (informative) Examples of quality criteria for selected MPS platforms and
applications .12
Annex C (informative) Reference material list .14
Bibliography .15
iv
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
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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
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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.
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.
v
Introduction
Massively parallel sequencing (MPS) is a high throughput analytical technology for nucleic acid
sequencing. MPS methods can process thousands to billions of nucleotide sequence reads simultaneously
in a single run, allowing whole genomes, transcriptomes and specific nucleic acid targets from different
organisms to be analysed in a relatively short time.
MPS is used in many life science disciplines permitting determination and high throughput analysis of
millions of nucleotide bases. The biological variability of deoxyribonucleic and ribonucleic acid polymers
from living organisms provides challenges in accurately determining their sequences. The quality of
sequence determination by MPS depends on many factors including, but not limited to, sample quality,
library preparation, and sequencing data quality.
The quality of nucleic acids and libraries prepared for MPS is critical to obtaining high quality sequence
data. Controlling the upstream processing steps of MPS and evaluating nucleic acid samples and
libraries for their suitability for sequencing significantly improves MPS results, downstream analyses
and ultimately conclusions dependent upon the MPS data.
vi
INTERNATIONAL STANDARD ISO 20397-1:2022(E)
Biotechnology — Massively parallel sequencing —
Part 1:
Nucleic acid and library preparation
1 Scope
This document specifies the general requirements for and gives guidance on quality assessments
of nucleic acid samples. It specifies general guidelines for library preparations and library quality
assessments prior to sequencing and data generation.
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 document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 20395:2019, Biotechnology — Requirements for evaluating the performance of quantification methods
for nucleic acid target sequences — qPCR and dPCR
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 20395:2019 and the following
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 https:// www .electropedia .org/
3.1
adapter
oligonucleotides of known sequence that are enzymatically added (e.g. ligase or polymerase chain
reaction) to the end(s) of a DNA/cDNA fragment
3.2
barcode
index
short sequence of typically six or more nucleotides that serve as a way to identify or label individual
samples when they are sequenced in parallel on a single sequencing lane, chip or both
Note 1 to entry: Barcodes are typically located within the sequencing adapters (3.1).
3.3
barcoding
indexing
unique DNA sequence identification
method that enables multiple samples to be pooled for sequencing
Note 1 to entry: Each sample is identified by a unique barcode (3.2), which enables identification of results during
the parallel analysis.
3.4
GC content
GC
percentage of guanine and cytosine in one or more nucleic acid sequence(s)
Note 1 to entry: The amount of guanine and cytosine in a polynucleotide is usually expressed as fraction (or
percentage) of total nitrogenous bases. Total nitrogenous bases comprise the total number of nucleotide bases of
reads from one or more MPS run.
[SOURCE: ISO 20397-2:2021, 3.15]
3.5
library
sequencing library
DNA, cDNA or RNA that has been prepared for massively parallel sequencing within a specific size range
and typically containing adapters (3.1) and/or identifiers recognised for sequence specific priming,
sequence capture, and/or identification of specific extracts
Note 1 to entry: Libraries can be DNA or cDNA. cDNA libraries are prepared for RNA sequencing on most
sequencers. Some instruments can directly sequence RNA.
3.6
library preparation
sequencing library preparation
set of procedures used to prepare DNA or RNA fragments containing tags, and sequencing primer
binding regions for massively parallel sequencing (MPS)
3.7
spike-in control
spike-in process control
target sequence often of defined sequence identity and concentration that are spiked in the sample at
various steps of the massively parallel sequencing protocol
Note 1 to entry: Process controls can be used to evaluate any protocol step but are typically applied as nucleic
acids controls prior to library preparation (3.6).
3.8
Q score
measure of the sequencing quality of a given nucleotide base
[SOURCE: ISO 20397-2:2021, 3.32, modified — The notes to entry have been deleted.]
4 Nucleic acid sample quality evaluation
4.1 General
The laboratory shall establish, implement and document a workflow for nucleic acid quantification that
ensures accurate and reproducible results. Requirements for nucleic acid sample quantity and quality
can vary between MPS methods. Nucleic acid purification methods can also affect the quality of nucleic
acids used for library preparation.
A quality control procedure shall be developed to clearly define nucleic acid quality and library
composition. This procedure shall be verified, implemented and documented, and permit accurate
quantification of nucleic acid at the minimum amount of nucleic acid required for the MPS performed.
The measurement uncertainty and sensitivity of the procedure used for this determination shall be
determined. The quantification allows appropriate adjustment of the nucleic acid concentration for
input into the MPS sequencer.
Annex A provides a quality control checklist. Quantity, purity and integrity are major quality indicators
for the prepared samples. Additional general considerations of sample quality regarding multiplex
molecular testing including NGS are available in ISO 21474-1:2020.
4.2 Sample quantification
A range of methods for nucleic acid quantification are provided in ISO 20395:2019, 5.2. Other methods
(e.g. electrophoresis) can also be used for the quantification.
Optimal sample amounts and concentrations appropriate for different MPS applications are listed in
Table B.2.
4.3 Sample purity
Nucleic acid sample purity analysis shall be conducted in accordance with methods specified in
ISO 20395:2019, 5.4.
4.4 Sample integrity
4.4.1 General
A range of methods used for assessing sample integrity is described in ISO 20395:2019, Annex B.
Gel electrophoresis and microfluidic analysis system can be used to evaluate nucleic acid sample
integrity.
4.4.2 Agarose gel electrophoresis
Agarose gel electrophoresis can be used as a method for separating and isolating different sized nucleic
acid molecules. It can also be used to determine nucleotide acid integrity. For example, optimally,
1)
genomic DNA (gDNA) samples have a strong main band of high molecular mass (greater than 20 kbp
in size) with minimal band dispersion.
4.4.3 Capillary gel electrophoresis
Capillary gel electrophoresis can also be used to assess nucleic acid integrity.
4.4.4 Microfluidic analysis system
Microfluidic analysis system can be used to assess the integrity of genomic DNA or RNA extracted from
various materials.
NOTE 1 DNA or RNA integrity number is commonly used as a numerical quality assessment criterion. The
higher the value, the better the quality.
NOTE 2 Specifies the appropriate threshold depending on the type of devices.
4.4.5 PCR method
A PCR method can be used for integrity evaluation. High quality samples can generate data that are
more useful than data generated from degraded samples.
NOTE Formalin-fixed and paraffin-embedded (FFPE) samples can cause challenges for some DNA
applications. Further guidance is given in the ISO 20166 series.
1) kbp = kilo base pairs.
5 Nucleic acid library preparation
5.1 General
The laboratory shall establish, implement and document each procedure for nucleic acid library
preparation that ensures accurate and reproducible results.
The quality of the MPS library is determined by the following procedures including, but not limited to:
a) fragmentation;
b) addition of universal sequences;
c) size selection;
d) amplification;
e) purification and clean up;
f) library quantification;
g) library qualification.
5.2 Fragmentation
5.2.1 General
Some sequencing methods (e.g. short read sequencing) require the template DNA, cDNA or RNA to be
fragmented as a first step prior to library preparation.
Fragmentation can be performed either mechanically or enzymatically to produce the DNA or RNA size
range that is required for the particular method and sequencing platform. Chemical fragmentation is
typically reserved for long RNA fragments.
The selection of a fragmentation method should take into account the impact of the specific approach
on evenness of coverage in the final libraries, e.g. to avoid the introduction of a GC bias.
The amount of starting material available and potential sample loss resulting from each approach
...