ISO/TC 276/WG 3 - Analytical methods

This document specifies general requirements and recommendations for quality assessments and control of massively parallel sequencing (MPS) data. It covers post raw data generation procedures, sequencing alignments, and variant calling. This document also gives general guidelines for validation and documentation of MPS data. This document does not apply to any processes related to de novo assembly.

This document specifies minimum requirements for the production and quality control of synthesized oligonucleotides (nominally up to 250 bases). This document also describes general quality attributes for synthesized oligonucleotides as well as common methods for evaluating quality attributes.

This document provides a method for evaluating aspects of the quality of a cell counting measurement process for a specific cell preparation through a set of quality indicators derived from a dilution series experimental design and statistical analysis. The quality indicators are based on repeatability of the measurement and the degree to which the results conform to an ideal proportional response to dilution. This method is applicable to total, differential, direct and indirect cell counting measurement processes, provided that the measurement process meets the criteria of the experimental design (e.g. cells are suspended in a solution). This method is most suitable during cell counting method development, optimization, validation, evaluation and/or verification of cell counting measurement processes. This method is especially applicable in cases where an appropriate reference material to assess accuracy is not readily available. This method does not directly provide the accuracy of the cell count. This method is primarily applicable to eukaryotic cells. NOTE Several sector/application specific international and national standards for cell counting exist. Where applicable, consulting existing standards when operating within their scope can be helpful.

This document provides generic requirements for evaluating the performance and ensuring the quality of methods used for the quantification of specific nucleic acid sequences (targets). This document is applicable to the quantification of DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) target sequences using either digital (dPCR) or quantitative real-time PCR (qPCR) amplification technologies. It applies to target sequences present in nucleic acid molecules including double-stranded DNA (dsDNA) such as genomic DNA (gDNA) and plasmid DNA, single stranded DNA (ssDNA), complementary DNA (cDNA), and single stranded RNA (ssRNA) including ribosomal RNA (rRNA), messenger RNA (mRNA), and long and short non-coding RNA [microRNAs (miRNAs) and short interfering RNAs (siRNAs)], as well as double-stranded RNA (dsRNA). This document applies to nucleic acids derived from biological sources such as viruses, prokaryotic and eukaryotic cells, cell-free biological fluids (e.g. plasma or cell media) or in vitro sources [e.g. oligonucleotides, synthetic gene constructs and in vitro transcribed (IVT) RNA]. This document is not applicable to quantification of very short DNA oligonucleotides ( This document covers: — analytical design including quantification strategies (nucleic acid copy number quantification using a calibration curve as in qPCR or through molecular counting as in dPCR, quantification relative to an independent sample and ratio measurements) and use of controls; — quantification of total nucleic acid mass concentration and quality control of a nucleic acid sample including assessment of nucleic acid quality (purity and integrity); — PCR assay design, optimization, in silico and in vitro specificity testing; — data quality control and analysis including acceptance criteria, threshold setting and normalization; — method validation (precision, linearity, limit of quantification, limit of detection, trueness and robustness) with specific requirements for qPCR and dPCR; — approaches to establishing metrological traceability and estimating measurement uncertainty. This document does not provide requirements or acceptance criteria for the sampling of biological materials or processing of biological samples (i.e. collection, preservation, transportation, storage, treatment and nucleic acid extraction). Nor does it provide requirements and acceptance criteria for specific applications (e.g. food or clinical applications where specific matrix issues can arise).

ISO 20391-1:2018 defines terms related to cell counting for biotechnology. It describes counting of cells in suspension (generally cell concentration) and cells adhered to a substrate (generally area density of cells). It provides key considerations for general counting methods (including total and differential counting, and direct and indirect counting) as well as for method selection, measurement process, and data analysis and reporting. ISO 20391-1:2018 is applicable to the counting of all cell types ? mammalian and non-mammalian (e.g. bacteria, yeast) cells. ISO 20391-1:2018 is not intended for counting of cells while in a tissue section or a biomaterial matrix. Several sector/application-specific international and national standards for cell counting currently exist. When applicable, the user can consult existing standards when operating within their scope (specific measurement techniques and/or applications).