Zika virus (ZIKV) is an arbovirus mainly transmitted through mosquito bites, but it can also be transmitted through sexual contact or vertical transmission from mother to child. It poses a great threat to pregnant women and can cause serious birth defects such as microcephaly in the fetus. At present, there are no targeted vaccines or specific drugs, making it urgent to screen and diagnose high-risk groups at an early stage. Nucleic acid testing plays a crucial role in the detection of Zika virus, and reference materials (RMs) are the basic standards for quantitative analysis of nucleic acid molecules. However, the lack of universal reference materials traceable to the International System of Units (SI) can affect the accuracy and comparability of quantitative results. In this study, we developed an in vitro transcribed (IVT) ZIKV NS1 gene RNA reference material (RM) and established an absolute quantification method based on one-step reverse transcription digital PCR (RT-dPCR) to characterize the RM. This method demonstrated excellent linearity across five orders of magnitude (10(0)-10(5) copies/mu L, R-2 > 0.9999). The accuracy of the RT-dPCR method was verified by isotope dilution mass spectrometry (IDMS), and there was no significant difference in copy number concentration between the two methods, with a reverse transcription efficiency value of 1.01. The consistency and reproducibility of the established RT-dPCR method were verified through measurements across different dPCR platforms and different laboratories. The prepared RM showed good homogeneity (F = 2.35 < F-0.05(10,F-11) = 2.85). Using the established RT-dPCR method, the reference value of the ZIKV RM was quantified as (1.41 +/- 0.08) & times; 10(5) copies/mu L, with an expanded uncertainty (coverage factor k = 2). The developed RM can provide a reliable standard for the quantitative detection of the ZIKV, improving accuracy and traceability in testing applications.

OBJECTIVES:This study developed a pseudoviral human rhinovirus (HRV) reference material using pseudovirus technology and integrating RT-dPCR with HPLC-IDMS to achieve accurate HRV RNA quantification, aiming to standardize HRV detection across labs and platforms. METHODS:We developed an RT-digital PCR (RT-dPCR) reference method for HRV, incorporating reverse transcription efficiency (RTE) correction using HPLC-isotope dilution mass spectrometry (HPLC-IDMS), which enables value assignment of the well-characterized pseudoviral reference material (RM) containing the conserved 5'UTR gene. RESULTS:The developed RT-dPCR method demonstrated high analytical sensitivity with a limit of detection (LoD) of eight copies/reaction and a limit of quantification (LoQ) of 11 copies/reaction. RTE was accurately determined as 101.25 % using HPLC-IDMS, enabling metrologically traceable RNA quantification. The pseudoviral RM was characterized using the traceable RT-dPCR method; its reference value and expanded uncertainty were determined to be (3.12 ± 0.69) × 103 copies/μL (k=2). The RM exhibited sufficient homogeneity and stability for 12 months at -80 °C, and its non-infectious nature coupled with its ability to simulate the entire workflow including nucleic acid extraction offers significant advantages. CONCLUSIONS:This study provided essential tools for standardizing HRV detection across different laboratories and platforms. This work enables traceable RNA quantification in SI units and simulates the full analytical process, thereby enhancing the accuracy and comparability of results. Furthermore, this approach serves as a transferable model for developing reference materials and traceable quantification methods for other respiratory pathogens, contributing to advancements in molecular diagnostics and public health.

RNA-sequencing's conversion of molecules to reads is inconsistent. Experiment-to-experiment variations (systemic bias) create batch effects, while gene-to-gene variations (sequence-dependent bias) invalidate inter-gene comparisons, precluding a universal scale. This confines analysis to relative fold-changes, a metric unreliable across batches. We introduce TranScale: 100 biomimetic standards with SI-traceable concentrations certified by Isotope Dilution Mass Spectrometry. Co-processed within samples, they empirically characterize systemic and sequence-dependent biases, generating a library-specific calibration curve (R² > 0.97) to convert reads into absolute quantities. This approach reveals that consistent fold-changes can mask severe absolute errors, exposing systemic biases missed by conventional QC. Across laboratories, this calibration reduced median inter-lab CV from >85% to <25% and increased biological signal-to-noise from ~0 to >7.9, outperforming the widely-used tool ComBat. By anchoring RNA-seq to the SI, our work establishes the metrological foundation for data interoperability and universal benchmarks, enabling absolute comparisons of SI-traceable quantities between any two genes.

The complete assembly of the human Y chromosome remains a challenge due to its highly repetitive and complex structure. While complete telomere-to-telomere (T2T) assemblies have been generated for a few individuals, such high-quality resources for East Asian populations, particularly for well-characterized multi-omics reference cohorts, are still scarce. The Chinese Quartet, comprising monozygotic twin daughters and their parents, is a premier reference material for genomic studies, yet a T2T-level Y chromosome assembly for this pedigree was lacking. Here, we present a complete, gapless T2T assembly of the Y chromosome (designated CQ-chrY) from the father of the Chinese Quartet. This assembly was generated by integrating Oxford Nanopore ultra-long reads, PacBio HiFi reads, and Hi-C data, resulting in a sequence of 61.88 Mb. The assembly shows exceptional base accuracy (QV = 51.09) and structural completeness (GCI = 100; CRAQ AQI = 95.217). We completely resolved the 33.52 Mb Yq12 heterochromatic region and annotated 164 protein-coding genes and 51.03 Mb (82.47%) of repetitive sequences. This CQ-chrY assembly represents the third complete Chinese Y chromosome and fills the last gap in the T2T assemblies of the Quartet family, providing an invaluable paternal haplotype resource for expanding East Asian genomic standards and for studies on Y chromosome structural variation and evolution.

The accurate detection and quantification of monkeypox virus (MPXV) is essential for effective viral diagnosis and epidemic control. However, there is currently a lack of high order reference methods and reference materials (RMs) for MPXV quantification, hindering consistent quality control across detection platforms. Complementing our previous work on the MPXV variant strain (F3L gene), this study establishes a novel reference measurement procedure (RMP) for the wild-type MPXV B6R gene and performs a comprehensive inter-laboratory validation for both targets. This method demonstrates excellent performance, with a broad dynamic range (22-23,802 copies/ reaction), strong linearity (R2 = 0.9985), and a low limit of quantitation (22 copies/reaction, CV <= 25%). Repeatability tests showed high precision, with inter-laboratory CVs consistently below 10% across nine different labs using various dPCR platforms. The recovery efficiencies for RMs of B6R and F3L genes were approximately 69% which was incorporated into the uncertainty budget. Homogeneity assessment revealed good consistency (CV = 2.94%), and stability studies confirmed sample stability at -70 degrees C and during short-term storage at 4 degrees C/ -20 degrees C. High inter-laboratory reproducibility of both wild-type and variant RMs was assessed by Mandel's statistical analysis and the Intraclass Correlation Coefficient (ICC). This validated dPCR RMP provides metrological traceability, providing reliable and comparable results for MPXV diagnosis and supporting public health efforts globally.

Sulfonamide resistance gene sul1 is widely distributed in the environment, and its accurate quantification serves as a core indicator for evaluating the level of antibiotic resistance pollution and the risk of resistance gene transmission. To ensure the comparability and reliability of monitoring data across different laboratories, the establishment of sul1 gene reference material (RM) is a fundamental prerequisite for achieving accurate measurement. In this study, the RM targeting sul1 gene was developed and quantified using both digital PCR (dPCR) and high-performance liquid chromatography-isotope dilution mass spectrometry (HPLC-IDMS). The established dPCR method exhibited good linearity (R-2 = 0.9999) and repeatability (RSD < 19%). For HPLC-IDMS, DNA was quantified by determining deoxynucleoside monophosphates (dNMPs) after enzymatic digestion, providing SItraceable quantification. There was no significant difference between the quantification results of the two methods, confirming good consistency. The prepared RM showed satisfactory homogeneity and stability under both long-term storage (-70( degrees)C) and short-term shipping conditions (on ice packs). The certified value of the RM was (1.32 +/- 0.10) & times; 10(9) copies/mu L (k = 2). This RM provides a traceable standard for quality control in sul1 detection, which will help improve the accuracy and comparability of antibiotic resistance gene quantification results.

Phred quality score (Q score) is critical for sequencing accuracy, yet the impact of Q40-achieving sequencing technologies (99.99

Multi-omics, as an incredibly useful technology, has been increasingly applied and enables new insights in life science and biomedical research. However, during the process of translating multi-omics technology into clinical applications, many issues remain, such as the irreproducibility of omics data, the incomparability of omics data from different platforms and laboratories, etc. The development of multi-omics measurement standards and reference materials can effectively improve the accuracy and reliability of multi-omics analysis and promote the rapid transformation of multi-omics technology into clinical applications. To address this issue, many international research consortiums and some National Metrology Institutes (NMIs) have begun to work on establishing reference materials (RM) for benchmarking multi-omics analysis. However, developing RMs for multi-omics faces significant challenges. Firstly, unlike traditional RMs, omics RMs have hundreds of thousands of reference values or even more, we cannot characterize the reference values one by one by establishing a primary reference measurement procedure for each measurand, especially since there is currently no such primary reference measurement method. Secondly, for the nominal properties, for example, variants in genomic DNA, how can we define these reference values and their related uncertainty or reliability. Finally, it is extremely difficult to obtain reproduceable absolute quantity in multi-omics at the current stage of technological development. The report will provide an overview of the research progress and challenge in multiple-omics RMs, with a focus on benchmarking germline variations in the human genome and multi-omics level. National Institute of Metrology (NIM) and Fudan University have jointly developed the Chinese quartet multi-omics RMs and its supporting standard reference dataset, which has been published in Nature Biotechnology and genome biology in 2023 [1,2,3]. We have successfully broken through the development mode of traditional RMs and establish the genomic DNA certified RMs with nominal properties and related reliabilities. We have proposed for the first time a ratio-based reference value for the quantitative omics, which is reproduceable on different platforms and laboratories, greatly eliminating batch effects and successfully obtained the certification for transcriptomics RMs from the State Administration for Market Regulation. These RMs are also used by the Clinical Laboratory Center of the National Health Commission in China to organize nationwide external quality assessment (EQA) for whole exon sequencing and transcriptomics sequencing, as well as to evaluate multi-omics performance across multi-center by the European EATRIS project. We will continuously update the reference datasets for genomic DNA and RNA based on new sequencing technology and user feedback. To improve the accuracy and reproducibility of cancer somatic variants calling , we are developing reference materials and reference datasets for paired tumor–normal cell lines. We hope these works can provide a benchmark for cancer somatic variants analysis to promote its clinical application.

The lack of quantitative methylation reference datasets (ground truth) and cross-laboratory reproducibility assessment hinders clinical translation of epigenome-wide sequencing technologies. Using certified Quartet DNA reference materials, here we generate 108 epigenome-sequencing datasets across three mainstream protocols (whole-genome bisulfite sequencing, enzymatic methyl-seq, and TET-assisted pyridine borane sequencing) with triplicates per sample across laboratories. We observe strand-specific methylation biases across all protocols and libraries. Cross-laboratory reproducibility analyses reveal high quantitative methylation levels agreement (mean Pearson correlation coefficient (PCC) = 0.96) but low detection concordance (mean Jaccard index = 0.36). Using consensus voting, we construct genome-wide quantitative methylation reference datasets serving as ground truth for proficiency testing. Key technical parameters-including mean CpG depth, coverage, and strand consistency-correlate strongly with reference-dependent quality metrics (recall, PCC, and RMSE). Collectively, these resources establish foundational standards for benchmarking emerging epigenomic technologies and analytical pipelines, enabling robust, standardized quality control in research and clinical applications.

The human norovirus (NV) is widely recognized as a pathogen causing acute gastroenteritis, particularly associated with elevated morbidity and mortality rates in pediatric and geriatric populations. While current detection methods, such as those based on immune reactions and quantitative polymerase chain reaction (qPCR), have been developed by researchers, these techniques do not provide absolute quantification. In this study, we established a reference material (RM) comprising pseudovirus particles containing NV ORF2 RNA. The RM was quantified using reverse transcription digital PCR (RT-dPCR), demonstrating ideal homogeneity and stability. The assigned value of the RM, including expanded uncertainty, was determined to be (3.42 +/- 0.69) x 103 copies/mu L. This RM can be effectively utilized to validate commercial diagnostic kits and platforms, thereby enhancing the accuracy and reliability of acute gastroenteritis diagnosis.

The genomic DNA reference material (RM) for human papillomavirus (HPV) plays a pivotal role in enhancing the standardization, accuracy, and reliability of HPV DNA testing, which is essential for global cervical cancer screening and evaluating the efficacy of HPV vaccination programs. This study presents the development of a stable C-33A cell line integrating the full-length HPV 45 genome, establishing a robust foundation for our RM. An absolute quantitative method based on digital droplet PCR (dPCR) was developed for characterizing this RM, demonstrating high accuracy and analytical sensitivity with a limit of detection (LoD) of 8.57 and limit of quantification (LoQ) of 16.8 copies per reaction. The homogeneity and stability of the RM were confirmed over 24 months at-80 degrees C, with a certified value and expanded uncertainty (U, k = 2) of (1.56 f 0.14) x 103 copies/ mu L. Evaluation of inter-laboratory reproducibility across five platforms validated the consistency of HPV 45 copy concentrations, with a coefficient of variation (CV) of less than 5.0 %. This work establishes metrological traceability for HPV 45, enhancing quality control in testing laboratories and providing a reliable standard for HPV DNA detection assays. Importantly, the methods and findings of this study are not only applicable to HPV 45 but also provide a blueprint for developing reference materials for other high-risk HPV types, potentially significantly advancing HPV diagnostics and screening globally. In conclusion, our study significantly advances HPV diagnostics by developing a RM (NIM-RM 5256) and a dPCR method suitable for global standardized assessment of HPV DNA detection, potentially improving cervical cancer screening programs and the management of HPV-related diseases.

In response to the demand for metrology in the context of the SARS-CoV-2 epidemic, National Institute of Metrology (NIM), China has undertaken a series of initiatives in the field of infectious disease testing, ranging from cutting-edge research to practical application. Three reference measurement procedures (RMP) published at JCTLM specify the operating specifications for SARS-CoV-2 (open reading frame 1ab, ORF1ab and nucleocapsid protein, N and Envelope, E gene) RNA copy number concentration by reverse transcription digital PCR (RT-dPCR). Certified Reference Materials including in vitro transcribed RNA, genomic RNA and Pseudoviral RNA of SARS-CoV-2 and different variants (GBW0928 to GBW09318) have been developed and widely applied for practical application. These reference materials were widely used by more than 1000 institutes or companies, including IVD manufacturers, CDCs, clinical laboratories, universities and institutes. Especially in helping many IVD manufacturers obtain certification for their testing kits from National Medical Products Administration of China (NMPA), FDA and CE, it has played an important role in providing metrological traceability. To investigate the international comparability in nucleic acid measurement and supporting CMC claim, NIM successfully coordinated two rounds’ comparisons for SARS-CoV-2 (CCQM-P199B and CCQM-K181) with LGC, NIBSC and NIST. Furthermore, to support other NMIs in APMP region to establish the capability in measurement of SARS-CoV-2, an international training course of “Accurate Measurement of SARS-CoV-2 and Development of Nucleic Acids Reference Material” was organized by NIM incorporated with HSA, KRSISS and NIST. Additionally, three rounds of proficiency testing for SARS-CoV-2 RNA and its different variants were conducted to enhance the capacity and guarantee the consistency of testing facilities in China. In 2023-2024, NIM has provided invaluable assistance to China CDC in the ongoing monitoring of SARS-CoV-2, particularly in the detection of the virus in urban sewage. This support includes providing simulated samples of Pseudoviral virus of SARS-CoV-2, as well as enriching the virus in urban sewage and employing highly accurate measurement methods. The whole framework can be used as the best practice of biometrology to support nucleic acids detection of infection disease and get prepared for future epidemic.

This study presents an advanced approach for the comprehensive analysis of low-abundance proteins in soybean seeds, addressing challenges posed by high-abundance storage proteins. We compared the effectiveness of Data-Dependent Acquisition (DDA), Data-Independent Acquisition (DIA), and BoxCar mass spectrometry techniques to identify low-abundance proteins in two types of soybean seeds: High-Oil and High-Protein seeds. The results indicate that the DIA method, and particularly the BoxCar methods, significantly improve the detection of low-abundance proteins compared to DDA, offering deeper insights into soybean seed biology. Specifically, BoxCar-based analysis revealed distinct proteomic differences between High-Oil and High-Protein seeds, highlighting more active metabolic processes in High-Oil seeds. Additionally, several key proteins were identified and annotated as uniquely expressed in either High-Oil or High-Protein seeds. These findings emphasize the importance of advanced proteomic techniques, such as BoxCar, in deepening our understanding of soybean seed biology and supporting breeding strategies to improve nutritional qualities.

Digital PCR (dPCR) technology is widely utilized for various applications, including the quantification of gene mutations and copy number variations. Certified reference materials (CRMs) play a critical role in improving the comparability of dPCR results, establishing SI-traceable copy number concentration values for dPCR calibration remains a key challenge due to the limited availability of CRMs value-assigned by higher-order, independent methods. To tackle this issue, a linearized plasmid DNA reference material (RM) was developed and rigorously characterized through an interlaboratory comparison involving three national measurement institutes (NMIs) from China (NIM), South Korea (KRISS), and Japan (NMIJ). The copy number concentration of the high-concentration RM was determined using two orthogonal methods: a dPCR-independent single molecule direct counting method and dPCR. Its homogeneity and stability were confirmed over the study duration. The equivalence of results among the NMIs was assessed using the En score, all En values were <1 indicating strong agreement and consistency with the candidate reference value within the expanded uncertainty. Additionally, a secondary CRM was generated by gravimetrically diluting the high-concentration RM to a nominal concentration of 104 copies/μL. This secondary CRM was used to evaluate four distinct dPCR platforms, revealing notable discrepancies in measurement results of up to 10.5 % among the platforms, potentially leading to substantial overestimation in nucleic acid quantification. These results emphasize the necessity of utilizing CRMs with SI-traceable certified values to validate dPCR quantification results and ensure comparability across different measurement systems.

The circulating blood proteome holds immense potential for biomarker discovery and understanding disease mechanisms. Notable advances in mass spectrometry and affinity-based technologies have been made, but data integration across studies and platforms is hindered by the absence of unified analytical standards. This limitation impedes comprehensive exploration of human biology across diverse phenotypes and cohorts as well as the translation of findings into clinical applications. The disparities between datasets, stemming from a combination of factors related to differences in sample collection, pre-analytical handling, measurement methods and instrumentation, further complicate data integration. In this Perspective, we outline key challenges in blood-based proteomics and propose actionable strategies. Central to our recommendations are high-quality, technology-agnostic reference samples, which can bridge disparate datasets and enable robust cross-study comparisons. By fostering interconnected investigations across proteomic technologies, blood sample collections, clinical phenotypes and different populations, these references will accelerate the field and its translation.

RNA reference materials and their corresponding reference datasets act as the ‘ground truth’ for the normalization of experimental values and are indispensable tools for reliably measuring intrinsically small differences in RNA-sequencing data, such as those between molecular subtypes of diseases in clinical samples. However, the variability in ‘absolute’ expression profiles measured across different batches, methods or platforms limits the use of conventional RNA reference datasets. We recently proposed a ratio-based method for constructing reference datasets. The ratio for a gene is defined as the normalized expression levels between two sample groups and produces more reliable values than the ‘absolute’ values obtained across diverse transcriptomic technologies and batches. Our gene ratios have been used for the successful generation of omics-wide reference datasets. Here, we describe a step-by-step process for establishing RNA reference materials and reference datasets, covering three stages: (1) reference materials, including material preparation, homogeneity testing and stability testing; (2) ratio-based reference datasets, including characterization, uncertainty estimation and orthogonal validation; and (3) applications, including definition of performance metrics, performing proficiency tests and diagnosing and correcting batch effects. This approach established the Quartet RNA reference materials and reference datasets (chinese-quartet.org) that have been approved as the first suite of nationally certified RNA reference materials by China’s State Administration for Market Regulation. The protocol can be utilized to establish and apply reference materials to improve RNA-sequencing data quality in diverse clinical settings. The procedure can be completed in 2 d and requires expertise in molecular biology and bioinformatics. The Quartet reference materials enable the integration and standardization of multiomics datasets acquired over a variety of instruments, samples and methods and the quantitative comparison of RNA sequencing data across laboratories.

The accurate detection of monkeypox virus (MPXV) is crucial for effective viral diagnosis and epidemic prevention. Currently, the field lacks standardized reference methods for MPXV quantification, as well as reliable pseudovirus reference materials (RMs) to ensure quality control throughout the detection process. To address this, we developed a droplet digital PCR (ddPCR) reference measurement procedure (RMP) for precise quantification of MPXV genes B6R , which demonstrates excellent performance with a wide dynamic range (11-1.24×104 copies/μL), strong linearity (R2=0.9984), and a low limit of quantitation (11 copies/μL, CV≤25%). Repeatability tests confirmed high precision, with inter-laboratory CVs <10% across nine labs using different dPCR platforms. recovery efficiencies for B6R and F3L were ∼69%, with uncertainties incorporated into final measurements. The homogeneity assessment showed good results (CV=2.94%), and stability studies confirmed the stability of the sample during -70 °C and short-term storage (4 °C/-20 °C). Mandel’s statistical data shows that the reproducibility between laboratories is consistent. This validated ddPCR RMP provides metrological traceability, ensuring reliable and comparable results for MPXV diagnosis and supporting public health efforts. ### Competing Interest Statement The authors have declared no competing interest. National Institute of Metrology, https://ror.org/05dw0p167, AKYZD2202

BACKGROUND/AIMS:Circulating tumor DNA (ctDNA) is becoming a valuable cancer biomarker for clinical decision-making. Nevertheless, the lack of quality control materials to assess the reliability of test results remains a challenge. This study aimed to establish digital PCR (dPCR) assays for detecting TP53 variants (R175H and R248W) and develop a preparation method for ctDNA reference materials to improve detection reliability. METHODS:Two dPCR assays targeting TP53-R175H and TP53-R248W variants were developed and validated for repeatability, sensitivity, and linearity. Additionally, a ctDNA reference material preparation protocol was developed by digesting nucleosomes from cultured cancer cell lines with micrococcal nuclease, followed by magnetic beads purification. The size distribution and quality of the generated ctDNA fragments was analyzed, and the developed dPCR assays were applied to detect the variants in the ctDNA samples. RESULTS:The dPCR assays demonstrated high repeatability (RSD of 0.16% to 7.65%) and excellent linearity (R2 values of 1.0000 and 0.9981) across variant allele frequencies of 50%-0.1%. The limits of detection (LOD) and quantification (LOQ) were 0.143% (R175H) and 0.092% (R248W). The ctDNA reference materials exhibited single dominant peaks at 128 bp (R175H) and 143 bp (R248W). The dPCR assays successfully detected variants in these reference materials, confirming their applicability for ctDNA samples. ONCLUSION:Firstly, accurate measurement procedures for TP53-R175H and TP53-R248W variants based on dPCR were established in this study. Furthermore, a protocol for preparing ctDNA reference material was established here. By digesting nucleosomal DNA derived from cancer cell lines with micrococcal nuclease, this method can closely mimic the properties of clinical ctDNA. The dPCR method and ctDNA reference material preparation approach established here could be used in ctDNA detection and for improving its reliability.

Epstein-Barr virus (EBV) is a distinct viral agent associated with a broad spectrum of diseases and has infected a significant proportion of the global population. Although EBV can remain latent throughout an individual’s lifetime, its reactivation plays a critical role in the pathogenesis of various diseases. A quantitative method was developed and applied to characterize the reference material (RM) containing the Epstein-Barr virus (EBV) genome. The assigned value, including expanded uncertainty, was determined to be (1529 ± 147) copies/μL. Validation studies of commercial kits revealed inconsistent performance levels, which may contribute to inaccurate diagnosis of EBV-related diseases. The integration of the quantitative method with the RM is anticipated to improve the accuracy and reliability of EBV detection and monitoring.