Foreword

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This document was prepared by Technical Committee ISO/TC 276 Biotechnology, Subcommittee SC 1, Analytical methods.

A list of all parts in the ISO 20397 series can be found on the ISO website.

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Introduction

Massively parallel sequencing (MPS) is a high-throughput analytical approach to nucleic acid sequencing utilizing massively parallel processing, that allows whole genomes, transcriptomes, and specific nucleic acid targets from different organisms to be investigated in a relatively short time.

Metagenomics approaches are an extremely powerful strategy in large-scale genomics applications as a way to study the taxonomic and functional composition of microbial communities from environmental, agricultural, and clinical primary samples/samples. Metagenomics does not require isolation of single bacterium from complex microbial community, but catalogues by sequencing all genes and genomes from total DNA (tDNA). It has great advantages to identify new species, including microorganisms that are difficult to culture under typical laboratory conditions.

Analysing metagenomics data involves a complex and statistically driven process that extends beyond traditional MPS pipelines to include identification, functional and relative abundance analyses. In metagenomics, whole genomic DNA is prepared from primary samples, regardless of its microbial composition and is characterized by whole genome sequencing. The annotation of resulting DNA fragments, individual reads or assembled sequence contigs, to individual taxonomic groups or known genome sequences, is carried out by sophisticated bioinformatic tools. The analysis is not limited to traditional MPS pipeline but also focuses on the identification of functional composition of a microbial community, which include the assignment of protein-coding open reading frames to functional categories, such as protein domain families or gene ontologies. Consequently, the analysis of whole genome sequencing (WGS) metagenomics data sets involves a significant statistical component, as sequence data must be evaluated based on relative abundances rather than on absolute presence/absence data.

1 Scope

This document specifies general requirements and guidance for metagenomics-dedicated sample preparation, and generating and analysing metagenomics sequence data obtained from massive parallel sequencing platforms. The specified metagenomics process includes the following stages:

sampling strategy and process, including type, storage, transportation, extraction, quality;
nucleic acid library preparation
design and review process including sequencing strategy and assessment;
database construction;
bioinformatics analysis and report
validation and verification for bioinformatics pipeline, and database

This document applies to laboratories and research organizations.

2 Normative references

3 Terms and definitions

4 Principle

4.1 General

5 Sampling strategy

5.1 General

5.2 Primary sample type

5.3 Primary sample stabilization and storage

5.4 Primary sample transportation

5.5 DNA/RNA isolation

5.6 DNA/RNA sample quality

6 Nucleic acid library preparation

7 Design and review process including sequencing strategy and assessment

7.1 General

7.2 Short read

7.3 Long read

7.4 Hybrid assembly

7.5 Sample preparation and library construction

7.6 Assessment

8 Database construction

8.1 General

8.2 Public database

8.3 Self-build database

9 Bioinformatics analysis

9.1 General

9.2 Identification list of microorganisms

10 Validation and verification

10.1 General

10.2 In silico sequence control for bioinformatics pipeline evaluation

10.3 Real sample control for pipeline evaluation

11 Evaluation

12 Test report

12.1 General

12.2 Test report content

Bibliography