ISO/TS 22692:2020

TECHNICAL ISO/TS
SPECIFICATION 22692
First edition
2020-10
Genomics informatic — Quality
control metrics for DNA sequencing
Informatique génomique — Mesures de contrôle de la qualité pour le
séquençage de l'ADN
Reference number
©
ISO 2020
© ISO 2020
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ii © ISO 2020 – All rights reserved

Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Abbreviated terms . 5
5 Quality control metrics for sample preparation . 5
5.1 General . 5
5.2 Sample sequencing type . 5
5.2.1 Sequencing type . 5
5.2.2 Target gene. 5
5.3 Sample information . 5
5.3.1 Specimen type . 5
5.3.2 Sampling date . 6
5.3.3 Specimen origin . 6
5.4 Summary of sample preparation related metrics . 6
6 Quality control metrics for library preparation . 6
6.1 General . 6
6.2 DNA extraction method . 6
6.2.1 DNA extraction kit . 6
6.3 DNA quality . 7
6.3.1 General. 7
6.3.2 DNA purity. 7
6.3.3 DNA integrity . 7
6.4 Library construction . 7
6.4.1 Library input amount . 7
6.4.2 Library insert size . 7
6.4.3 Library construction kit . 7
6.5 Summary of library preparation related metrics . 7
7 Quality control metrics for sequencing . 8
7.1 General . 8
7.2 Sequencing information . 8
7.2.1 Sequencing instrument . 8
7.2.2 Read length . 8
7.2.3 Sequencing direction . . 8
7.2.4 Running mode . 8
7.3 Running quality information . 9
7.3.1 Error rate . 9
7.3.2 Percent data quality >Q30 . 9
7.4 Summary of sequencing related metrics . 9
8 Quality control metrics for data processing . 9
8.1 General . 9
8.2 Data quality measurement . 9
8.2.1 Total reads . 9
8.2.2 Mean coverage . 9
8.2.3 Uniformity .10
8.2.4 Duplication rate .10
8.2.5 On-target rate .10
8.2.6 Q30 rate .10
8.3 Sequencing alignment.10
8.3.1 Mapping algorithm.10
8.3.2 Local realignment software and version .10
8.4 Variant calling .10
8.4.1 Variant calling software and version .10
8.4.2 Variant call quality score . .10
8.4.3 Allelic read percentage & ratio .10
8.5 Variant filtering and annotation.11
8.5.1 General.11
8.5.2 Germline filter criteria .11
8.5.3 Mutation and annotation database .11
8.6 Summary of data processing related metrics .11
Annex A (informative) Example layout of quality control metrics .12
Bibliography .14
iv © ISO 2020 – All rights reserved

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
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 215, Health informatics, Subcommittee
SC 1, Genomics informatics.
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.
Introduction
The rapid progress in Next Generation Sequencing (NGS) technology has drastically reduced the
cost and time for genomic analysis. A number of research institutions, corporations, and government
agencies are competitively collecting a large volume of genomic data through multi-national, multi-
[9] [10] [11]
institutional projects such as “DiscovEHR” , “gnomAD” and “UK Biobank” . The demand for
sharing of “high quality” genomic data is growing because large-scale reference data is required for
reliable detection of mutation for both industrial and clinical applications.
However, the quality of available genomic data is less than desirable. To establish consistent quality
control metrics, details of each stage of NGS process need to be recorded, shared and standardized
(processes and data elements collected and coded for each stage and sub-stage). These processes
include sample preparation, library preparation, sequencing, and data processing, among others, as
shown in Figure 1.
Figure 1 — NGS process
vi © ISO 2020 – All rights reserved

TECHNICAL SPECIFICATION ISO/TS 22692:2020(E)
Genomics informatic — Quality control metrics for DNA
sequencing
1 Scope
This document identifies quality metrics for the detection of DNA variants using next generation
sequencing (NGS) technology. It also defines the data types, relationships, optionality, cardinalities and
terminology bindings of the data.
This document provides a basis for sharing and for the application of “high quality” genomic data and
contributes to the realization of the precision medicine and the development of relevant industries.
This document is intended to serve as a catalogue of sequencing data elements used to address quality
metrics for various clinical, industrial and commercial applications. The exchange of these data allows
researchers, commercial entities, and regulatory bodies to assess for the purpose of selective utilization
of the data by setting application-specific quality criteria
This document is not intended for
— sequencing methods other than NGS, such as the Sanger sequencing,
— targets other than genome, such as transcriptome or proteome, or
— specimens of species other than humans.
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
copy number variation
CNV
variation (3.18) in the number of copies of one or more sections of the DNA (3.3)
[SOURCE: ISO/TS 20428:2017, 3.7]
3.2
deletion
contiguous removal of one or more bases from a genomic sequence
[SOURCE: ISO/IEC 23092-2:2019, 3.4]
3.3
DNA
deoxyribonucleic acid
molecule that exists in nuclei and in mitochondria of human cells and is composed of a linear array of 4
bases (Adenine: A, Thymine: T, Guanine: G and Cytosine: C)
[SOURCE: ISO 18074:2015, 4.1, modified — Note 1 to entry deleted.]
3.4
DNA sequencing
determining the order of nucleotide bases (adenine, guanine, cytosine and thymine) in a molecule of
DNA (3.3)
Note 1 to entry: Sequence is generally described from the 5’ end.
[SOURCE: ISO/TS 17822-1:2014, 3.20]
3.5
exome
part of the genome formed by exons
[SOURCE: ISO/TS 20428:2017, 3.13]
3.6
FASTA
genomic information representation that includes a name and a nucleotide sequence for each sequence
read (3.17)
[SOURCE: ISO/IEC 23092-2:2019, 3.7, modified]
3.7
FASTQ
genomic information representation that includes FASTA (3.6) and quality values
[SOURCE: ISO/IEC 23092-2:2019, 3.8]
3.8
gene
basic unit of hereditary information composed of chains of nucleotide base pairs in specific sequences
that encodes a protein or protein subunit
[SOURCE: ISO 11238:2018, 3.29]
3.9
germline
series of germ cells each descended or developed from earlier cells in the series, regarded as continuing
through successive generations of an organism
[SOURCE: ISO/TS 20428:2017, 3.17]
3.10
indel
insertion (3.11) or/and deletion (3.2)
[SOURCE: ISO/TS 20428:2017, 3.18]
3.11
insertion
contiguous addition of one or more bases into a genomic sequence
[SOURCE: ISO/IEC 23092-2:2019, 3.18]
2 © ISO 2020 – All rights reserved

3.12
large indel
insertion (3.11) or deletion (3.2) up to around 1 kb
[SOURCE: ISO/TS 20428:2017, 3.21]
3.13
nucleotide
monomer of a nucleic acid polymer such as DNA (3.3) or RNA
Note 1 to entry: Nucleotides are denoted as letters (‘A’ for adenine; ‘C’ for cytosine; ‘G’ for guanine; ‘T’ for thymine
which only occurs in DNA; and ‘U’ for uracil which only occurs in RNA). The chemical formula for a specific
DNA or RNA molecule is given by the sequence of its nucleotides, which can be represented as a string over the
alphabet (‘A’, ’C’, ’G’, ‘T’) in the case of DNA, and a string over the alphabet (‘A’, ‘C’, ‘G’, ‘U’) in the case of RNA. Bases
with unknown molecular composition are denoted with ‘N’.
[SOURCE: ISO/IEC 23092-2:2019, 3.20]
3.14
polymerase chain reaction
PCR
in vitro enzymatic technique to increase the number of copies of a specific DNA fragment by several
orders of magnitude
[SOURCE: ISO 16577:2016, 3.148]
3.15
quality score
Phred quality score
Q score
quality measure used to assess the accuracy of a sequencing reaction
Note 1 to entry: This quality measure indicates the probability that a given base is called incorrectly by the
sequencer. Phred scores are on a logarithmic scale. Therefore, if Phred assigns a Q score of 30 (Q30) to a base, this
is equivalent to the probability of an incorrect base call 1 in 1 000 times. A lower base call accuracy of 99 % (Q20)
will have an incorrect base call probability of 1 in 100, meaning that every 100 base pairs sequencing read will
likely contain an error.
[SOURCE: ISO 21286:2019, 3.4]
3.16
reference sequence
nucleic acid sequence with biological relevance
Note 1 to entry: Each reference sequence is indexed by a one-dimensional integer coordinate system whereby
each integer within range identifies a single nucleotide. Coordinate values can only be equal to or larger than
zero. The coordinate system in the context of this standard is zero-based (i.e. the first nucleotide has coordinate
0 and it is said to be at position 0) and linearly increasing within the string from left to right.
[SOURCE: ISO/IEC 23092-1:2019, 3.22]
3.17
sequence read
read
fragmented nucleotide sequences that are used to reconstruct the original sequence for next generation
sequencing technologies
[SOURCE: ISO/TS 20428:2017, 3.26]
3.18
sequence variation
DNA sequence variation
variation
differences of DNA sequence among individuals in a population
Note 1 to entry: Variation implies copy number variation (3.1), deletion (3.2), insertion (3.11), indel (3.10), small
indel (3.20), large indel (3.12), or single nucleotide variant (3.19).
[SOURCE: ISO/TS 20428:2017, 3.30]
3.19
single nucleotide variant
SNV
DNA sequence variation (3.18) that occurs when a single nucleotide, A, T, C, or G, in the genome (or other
target sequence) differs between templates
[SOURCE: ISO 20395:2019, 3.35]
3.20
small indel
insertion (3.11) or deletion (3.2) of 2 nucleotides to 100 nucleotides
[SOURCE: ISO/TS 20428:2017, 3.32]
3.21
specimen
biospecimen
biological specimen
sample of tissue, body fluid, food, or other substance that is collected or acquired to support the
assessment, diagnosis, treatment, mitigation or prevention of a disease, disorder or abnormal physical
state, or its symptoms
[SOURCE: ISO/TS 20428:2017, 3.34]
3.22
targeted sequencing
disease-targeted gene panel
technique used for sequencing only selected/targeted genomic regions of interest from a DNA sample
Note 1 to entry: For further details, see Reference [12].
3.23
whole exome sequencing
WES
technique for sequencing the exomes (3.5) of the protein-coding genes (3.8) in a genome
[SOURCE: ISO/TS 20428:2017, 3.38]
3.24
whole genome sequencing
WGS
technique that determines the complete DNA sequence of an organism’s genome at a single time
[SOURCE: ISO/TS 20428:2017, 3.39]
4 © ISO 2020 – All rights reserved

4 Abbreviated terms
BAM Binary Alignment/Map
BED Browser Extensible Data
FMA Foundational Model of Anatomy
HGNC HUGO Gene Nomenclature Committee
HUGO Human
...