ISO/PRF TS 22692

TECHNICAL ISO/TS
SPECIFICATION 22692
First edition
Genomics informatic — Quality
control metrics for DNA sequencing
PROOF/ÉPREUVE
Reference number
ISO/TS 22692:2020(E)
ISO 2020
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ISO/TS 22692:2020(E)
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© ISO 2020

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

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

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

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

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

4 Abbreviated terms .............................................................................................................................................................................................. 4

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 ..................................................................................................................................................................... 5

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 ................................................................................................................................................................................................ 6

6.3.1 General...................................................................................................................................................................................... 6

6.3.2 DNA purity............................................................................................................................................................................. 6

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 ........................................................................................................................................ 7

7.1 General ........................................................................................................................................................................................................... 7

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 ...................................................................................................................................................... 8

7.3.1 Error rate ................................................................................................................................................................................ 8

7.3.2 Percent data quality >Q30 ....................................................................................................................................... 8

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 ............................................................................................................................................................................. 9

8.2.4 Duplication rate ................................................................................................................................................................ 9

8.2.5 On-target rate ..................................................................................................................................................................... 9

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............................................................................................................................................10

8.5.1 General...................................................................................................................................................................................10

8.5.2 Germline filter criteria .............................................................................................................................................10

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

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This document was prepared by Technical Committee ISO/TC 215, Health informatics, Subcommittee SC

Any feedback or questions on this document should be directed to the user’s national standards body. A

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-

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

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

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

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

variation (3.18) in the number of copies of one or more sections of the DNA (3.3)

molecule that exists in nuclei and in mitochondria of human cells and is composed of a linear array of 4

determining the order of nucleotide bases (adenine, guanine, cytosine and thymine) in a molecule of

genomic information representation that includes a name and a nucleotide sequence for each sequence

basic unit of hereditary information composed of chains of nucleotide base pairs in specific sequences

series of germ cells each descended or developed from earlier cells in the series, regarded as continuing

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

in vitro enzymatic technique to increase the number of copies of a specific DNA fragment by several

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

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.

fragmented nucleotide sequences that are used to reconstruct the original sequence for next generation

Note 1 to entry: Variation implies copy number variation (3.1), deletion (3.2), insertion (3.11), indel (3.10), small

DNA sequence variation (3.18) that occurs when a single nucleotide, A, T, C, or G, in the genome (or other

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

technique used for sequencing only selected/targeted genomic regions of interest from a DNA sample

technique for sequencing the exomes (3.5) of the protein-coding genes (3.8) in a genome

technique that determines the complete DNA sequence of an organism’s genome at a single time

As the first step in NGS workflow, sample preparation is the process to assess and review the submitted

sample before being accepted for sequencing. The amount of information in sequencing data is quite

different according to sequencing types (whole genome sequencing, whole exome sequencing, and

targeted sequencing) and specimen types (blood, surgical tissue, biopsy, etc.), so quality control metrics

for sample preparation would be used to select appropriate comparison data in analysis process.

Sequencing types are three levels of analysis via NGS: WGS, WES, and targeted sequencing . WGS

covers all regions of the genome: both coding and noncoding regions. WES covers all coding regions,

which is estimated to comprise 1% to 2 % of the genome, yet contains around 85 % of recognized

disease-related mutations . Targeted sequencing interrogates known disease-associated genes,

Sets of genes or gene regions for targeted sequencing. Target gene is represented in accordance with

Type of specimen (e.g. whole blood, cell, urine, fresh cell & tissue) with related data during sample

collection (e.g. biopsy, surgical excision, EDTA, heparin), processing (e.g. formalin, centrifugation),

and storage (e.g. paraffin block, cryotube). Specimen type is represented in accordance with SPREC of

Sampling date is the date when the specimen was acquired. It is represented in accordance with the

Specimen origin refers to the anatomical site from which the specimen was acquired. The anatomical

EXAMPLE SNOMED CT® fully specified name: Lung structure (body structure), concept identifier:

The summary of quality control metrics for sample preparation shall be in accordance with Table 1.

Library preparation is the process of generating DNA fragments to meet the input requirement of

sequencing platforms. In general, this workflow involves DNA extraction, DNA quality measurement

and library construction. Depending on the purposes of sequencing, library construction methods can

vary and sequencing results will be affected. Therefore, for quality control of the data, the conditions

The ratio of optical density (OD 260/280, OD 260/230) is used as an indicator of the sample purity.

1) SNOMED CT® is the registered trademark of the International Health Terminology Standards Development

Organisation (IHTSDO). This information is given for the convenience of users of this document and does not

The absence of both single or double strand breaks and absence of nucleotide modifications in the

NOTE Agilent® 2200 TapeStation® provides a numerical assessment of gDNA integrity referred as the

Library insert size used to construct the library, which can be confirmed by quality control at the

Product, product’s version and manufacturer’s name of the library construction kit.

The summary of quality control metrics for library preparation shall be in accordance with Table 2.