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

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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 20397-1:2022(E)
© ISO 2022
---------------------- Page: 1 ----------------------
ISO 20397-1:2022(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: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
© ISO 2022 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 20397-1:2022(E)
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
© ISO 2022 – All rights reserved
---------------------- Page: 3 ----------------------
ISO 20397-1:2022(E)

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

© ISO 2022 – All rights reserved
---------------------- Page: 4 ----------------------
ISO 20397-1:2022(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 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.
© ISO 2022 – All rights reserved
---------------------- Page: 5 ----------------------
ISO 20397-1:2022(E)
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.
© ISO 2022 – All rights reserved
---------------------- Page: 6 ----------------------
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.
© ISO 2022 – All rights reserved
---------------------- Page: 7 ----------------------
ISO 20397-1:2022(E)
3.4
GC content
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.
© ISO 2022 – All rights reserved
---------------------- Page: 8 ----------------------
ISO 20397-1:2022(E)

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,

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.
© ISO 2022 – All rights reserved
---------------------- Page: 9 ----------------------
ISO 20397-1:2022(E)
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

should also be considered.
5.2.2 Mechanical fragmentation

Mechanical shearing can be performed using focused acoustic shearing devices. The resulting fragment

sizes (150 bp to 5 000 bp) can be controlled by varying the intensity and duration of ultrasonic

acoustic waves.

Hydrodynamic shearing can be used to produce larger fragments (typically 1 kbp to 75 kbp), but

requires large DNA input amounts (>1 µg) and the throughput is low.

Nebulization is another alternative, which uses compressed air to force DNA or RNA through a small

hole. Although fragment size can be controlled to an extent, large amounts of input DNA (microgram

quantities) are required and the method is only suitable for small sample sizes.
5.2.3 Enzymatic fragmentation

Enzymatic fragmentation methods (e.g. using fragmentases, transposases or endonucleases) have

the advantage of higher throughput compared to mechanical methods and typically result in a lower

2) bp = base pairs.
© ISO 2022 – All rights reserved
---------------------- Page: 10 ----------------------
ISO 20397-1:2022(E)

sample loss. The disadvantage of enzymatic approaches is that they can typically result in sequence

bias since many enzymes have specific recognition sequences or sequence preferences.

5.2.4 Chemical fragmentation

Chemical fragmentation is typically reserved for breaking up long RNA fragments. Chemical

fragmentation is performed by heating RNA with a divalent metal cation (magnesium or zinc). The

length of the resulting products ranges from 115 base nucleotides to 350 base nucleotides and can be

adjusted by increasing or decreasing the time of incubation.
5.2.5 Fragmented nucleic acid sample quantity

Certain library preparation protocols require the fragmented DNA or RNA to be quantified (as some

material can be lost during the process). This can be performed using any of the methods described in

4.2, but most typically this is done with spectrophotometry or intercalating fluorescent dyes.

5.2.6 Fragmented nucleic acid sample purity

If there is a risk that impurities from the fragmentation process (e.g. components of enzymatic

fragmentation reactions) can be carried over into the purified products, the sample purity can be

assessed using the methods described in 4.3.
5.2.7 Fragmented nucleic acid size distribution

The fragmented nucleic acid should be checked to determine whether the appropriate size range has

been achieved. This can be done using the methods described for monitoring sample integrity in 4.4.

5.2.8 Fragmented nucleic acid purification using gel electrophoresis

Purification of fragmented nucleic acids can be done by separating nucleic acids with a specific size from

those with other sizes before downstream library preparation and sequencing steps. The purification

can be achieved by using capillary electrophoresis, bead-based methods or other electrophoresis

methods. The purified nucleic acids can be quantified as described in 4.2.
5.3 Addition of universal sequences
5.3.1 Repair

Because damage can rise from fragmentation, the nucleic acid sample shall be repaired after this

process to improve efficiency in subsequent preparation steps.

NOTE The following conditions can warrant a repair procedure: abasic sites, nicks, thymine dimers, blocked

3′-ends, oxidized guanines or pyrimidines, deaminated cytosine.

The end of the fragment shall be polished (i.e. the addition of 5′-PO or 3′-OH) to make it suitable for

ligation.
5.3.2 Ligation of adapter
Evaluation of the adapter sequence can include, but is not limited to:
a) length;
b) the design of the adapter;
c) the ratio of adapter to DNA.
The ratio of adapter is critical and requires optimization.
© ISO 2022 – All rights reserved
---------------------- Page: 11 ----------------------
ISO 20397-1:2022(E)
5.3.3 Barcoding/indexing

Libraries can be single or dual indexed. Unique dual indexing can be used with MPS platforms that use

patterned flow cells to mitigate the effects of incorrect assignment of libraries from the expected index

to a different index.
The sequence of barcodes set should be as unique and heterologous as possible.

Nucleotide sequences chosen for barcodes/indexes should be differentiable within the sequencing

project.

Barcode length should be as short as possible. See Table B.1 for example references for this quality

metric.
The barcode should be designed to minimize adapter-dimer/primer-dimer artefacts.

The sequence of the barcode/index normally consists of a 6 bp to 12 bp. Each barcode/index in a DNA

sequencing library should have a unique sequence that is easily differentiable from all of the other

barcodes/indexes in that DNA sequencing library.
Barcodes are normally located next to the adapters.
The set of barcode sequences can be evaluated using the following parameters:
a) diversity of barcode sequences (relevant to some MPS platforms);
b) hamming distance (number of bases difference between each index combination).
If b) is used for the evaluation, the hammering distance should be > 3 bp.
5.4 Size selection

The optimal length of nucleotides in the library should be determined for the specific application. This

is typically achieved by using the following methods including, but not limited to:

a) bead-based methods;
b) electrophoresis.
5.5 Amplification
Bias of polymerase can cause a failure of the amplification.

Polymerase can introduce errors; this can be minimized by increasing the sequence coverage or

technical replicates.
NOTE 1 Overamplification produces PCR duplicates.
NOTE 2 High-fidelity amplification enzyme can be used to reduce errors.
5.6 Purification and clean up procedures

Before MPS is performed, the PCR-amplified pool of fragments or unfragmented nucleic acid should be

cleaned to remove excess adapters or other contaminants. Techniques can include, but are not limited

to:
a) bead clean up;
b) spin column;
c) en
...

FINAL
INTERNATIONAL ISO/FDIS
DRAFT
STANDARD 20397-1
ISO/TC 276
Biotechnology — Massively parallel
Secretariat: DIN
sequencing —
Voting begins on:
2021-12-22
Part 1:
Voting terminates on:
Nucleic acid and library preparation
2022-02-16
RECIPIENTS OF THIS DRAFT ARE INVITED TO
SUBMIT, WITH THEIR COMMENTS, NOTIFICATION
OF ANY RELEVANT PATENT RIGHTS OF WHICH
THEY ARE AWARE AND TO PROVIDE SUPPOR TING
DOCUMENTATION.
IN ADDITION TO THEIR EVALUATION AS
Reference number
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO-
ISO/FDIS 20397-1:2021(E)
LOGICAL, COMMERCIAL AND USER PURPOSES,
DRAFT INTERNATIONAL STANDARDS MAY ON
OCCASION HAVE TO BE CONSIDERED IN THE
LIGHT OF THEIR POTENTIAL TO BECOME STAN-
DARDS TO WHICH REFERENCE MAY BE MADE IN
NATIONAL REGULATIONS. © ISO 2021
---------------------- Page: 1 ----------------------
ISO/FDIS 20397-1:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on

the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below

or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
© ISO 2021 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/FDIS 20397-1:2021(E)
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
© ISO 2021 – All rights reserved
---------------------- Page: 3 ----------------------
ISO/FDIS 20397-1:2021(E)

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

© ISO 2021 – All rights reserved
---------------------- Page: 4 ----------------------
ISO/FDIS 20397-1:2021(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 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.
© ISO 2021 – All rights reserved
---------------------- Page: 5 ----------------------
ISO/FDIS 20397-1:2021(E)
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.
© ISO 2021 – All rights reserved
---------------------- Page: 6 ----------------------
FINAL DRAFT INTERNATIONAL STANDARD ISO/FDIS 20397-1:2021(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.
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3.4
GC content
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.
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ISO/FDIS 20397-1:2021(E)

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,

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.
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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

should also be considered.
5.2.2 Mechanical fragmentation

Mechanical shearing can be performed using focused acoustic shearing devices. The resulting fragment

sizes (150 bp to 5 000 bp) can be controlled by varying the intensity and duration of ultrasonic

acoustic waves.

Hydrodynamic shearing can be used to produce larger fragments (typically 1 kbp to 75 kbp), but

requires large DNA input amounts (>1 µg) and the throughput is low.

Nebulization is another alternative, which uses compressed air to force DNA or RNA through a small

hole. Although fragment size can be controlled to an extent, large amounts of input DNA (microgram

quantities) are required and the method is only suitable for small sample sizes.
5.2.3 Enzymatic fragmentation

Enzymatic fragmentation methods (e.g. using fragmentases, transposases or endonucleases) have

the advantage of higher throughput compared to mechanical methods and typically result in a lower

2) bp = base pairs.
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sample loss. The disadvantage of enzymatic approaches is that they can typically result in sequence

bias since many enzymes have specific recognition sequences or sequence preferences.

5.2.4 Chemical fragmentation

Chemical fragmentation is typically reserved for breaking up long RNA fragments. Chemical

fragmentation is performed by heating RNA with a divalent metal cation (magnesium or zinc). The

length of the resulting products ranges from 115 base nucleotides to 350 base nucleotides and can be

adjusted by increasing or decreasing the time of incubation.
5.2.5 Fragmented nucleic acid sample quantity

Certain library preparation protocols require the fragmented DNA or RNA to be quantified (as some

material can be lost during the process). This can be performed using any of the methods described in

4.2, but most typically this is done with spectrophotometry or intercalating fluorescent dyes.

5.2.6 Fragmented nucleic acid sample purity

If there is a risk that impurities from the fragmentation process (e.g. components of enzymatic

fragmentation reactions) can be carried over into the purified products, the sample purity can be

assessed using the methods described in 4.3.
5.2.7 Fragmented nucleic acid size distribution

The fragmented nucleic acid should be checked to determine whether the appropriate size range has

been achieved. This can be done using the methods described for monitoring sample integrity in 4.4.

5.2.8 Fragmented nucleic acid purification using gel electrophoresis

Purification of fragmented nucleic acids can be done by separating nucleic acids with a specific size from

those with other sizes before downstream library preparation and sequencing steps. The purification

can be achieved by using capillary electrophoresis, bead-based methods or other electrophoresis

methods. The purified nucleic acids can be quantified as described in 4.2.
5.3 Addition of universal sequences
5.3.1 Repair

Because damage can rise from fragmentation, the nucleic acid sample shall be repaired after this

process to improve efficiency in subsequent preparation steps.

NOTE The following conditions can warrant a repair procedure: abasic sites, nicks, thymine dimers, blocked

3′-ends, oxidized guanines or pyrimidines, deaminated cytosine.

The end of the fragment shall be polished (i.e. the addition of 5′-PO or 3′-OH) to make it suitable for

ligation.
5.3.2 Ligation of adapter
Evaluation of the adapter sequence can include, but is not limited to:
a) length;
b) the design of the adapter;
c) the ratio of adapter to DNA.
The ratio of adapter is critical and requires optimization.
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5.3.3 Barcoding/indexing

Libraries can be single or dual indexed. Unique dual indexing can be used with MPS platforms that use

patterned flow cells to mitigate the effects of incorrect assignment of libraries from the expected index

to a different index.
The sequence of barcodes set should be as unique and heterologous as possible.

Nucleotide sequences chosen for barcodes/indexes should be differentiable within the sequencing

project.

Barcode length should be as short as possible. See Table B.1 for example references for this quality

metric.
The barcode should be designed to minimize adapter-dimer/primer-dimer artefacts.

The sequence of the barcode/index normally consists of a 6 bp to 12 bp. Each barcode/index in a DNA

sequencing library should have a unique sequence that is easily differentiable from all of the other

barcodes/indexes in that DNA sequencing library.
Barcodes are normally located next to the adapters.
The set of barcode sequences can be evaluated using the following parameters:
a) diversity of barcode sequences (relevant to some MPS platforms);
b) hamming distance (number of bases difference between each index combination).
If b) is used for the evaluation, the hammering distance should be > 3 bp.
5.4 Size selection

The optimal length of nucleotides in the library should be determined for the specific application. This

is typically achieved by using the following methods including, but not limited to:

a) bead-based methods;
b) electrophoresis.
5.5 Amplification
Bias of polymerase can cause a failure of the amplifi
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