Differential abundance analysis is controversial throughout microbiome research. Gold standard approaches require laborious measurements of total microbial load, or absolute number of microorganisms, to accurately determine taxonomic shifts. Therefore, most studies rely on relative abundance data. Here, we demonstrate common pitfalls in comparing relative abundance across samples and identify two solutions that reveal microbial changes without the need to estimate total microbial load. We define the notion of "reference frames", which provide deep intuition about the compositional nature of microbiome data. In an oral time series experiment, reference frames alleviate false positives and produce consistent results on both raw and cell-count normalized data. Furthermore, reference frames identify consistent, differentially abundant microbes previously undetected in two independent published datasets from subjects with atopic dermatitis. These methods allow reassessment of published relative abundance data to reveal reproducible microbial changes from standard sequencing output without the need for new assays.

Detection of somatic mosaicism in non-proliferative cells is a new challenge in genome research, however, the accuracy of current detection strategies remains uncertain due to the lack of a ground truth. Herein, we sought to present a set of ultra-deep sequenced WES data based on reference standards generated by cell line mixtures, providing a total of 386,613 mosaic single-nucleotide variants (SNVs) and insertion-deletion mutations (INDELs) with variant allele frequencies (VAFs) ranging from 0.5% to 56%, as well as 35,113,417 non-variant and 19,936 germline variant sites as a negative control. The whole reference standard set mimics the cumulative aspect of mosaic variant acquisition such as in the early developmental stage owing to the progressive mixing of cell lines with established genotypes, ultimately unveiling 741 possible inter-sample relationships with respect to variant sharing and asymmetry in VAFs. We expect that our reference data will be essential for optimizing the current use of mosaic variant detection strategies and for developing algorithms to enable future improvements.

Tumor mutational burden (TMB), measured by exome or panel sequencing of tumor tissue or blood (bTMB), is a potential predictive biomarker for treatment benefit in patients with various cancer types receiving immunotherapy targeting checkpoint pathways. However, significant variability in TMB measurement has been observed. We developed contrived bTMB reference materials using DNA from tumor cell lines and donor-matched lymphoblastoid cell lines to support calibration and alignment across laboratories and platforms. Contrived bTMB reference materials were developed using genomic DNA from lung tumor cell lines blended into donor-matched lymphoblastoid cell lines at 0.5% and 2% tumor content, fragmented and size-selected to mirror the size profile of circulating cell-free tumor DNA with TMB scores of 7, 9, 20, and 26 mut/Mb. Variant allele frequency (VAF) and bTMB scores were assessed using PredicineATLAS and GuardantOMNI next-generation sequencing assays. DNA fragment sizes in the contrived reference samples were similar to those found within patient plasma-derived cell-free DNA, and mutational patterns aligned with those in the parental tumor lines. For the 7, 20, and 26 mut/Mb contrived reference samples with 2% tumor content, bTMB scores estimated using either assay aligned with expected scores from the parental tumor cell lines and showed good reproducibility. A bioinformatic filtration step was required to account for low-VAF artifact variants. We demonstrate the feasibility and challenges of producing and using bTMB reference standards across a range of bTMB levels, and how such standards could support the calibration and validation of bTMB platforms and help harmonization between panels and laboratories.

Quantitative fluorescence and superresolution microscopy are often limited by insufficient data quality or artifacts. In this context, it is essential to have biologically relevant control samples to benchmark and optimize the quality of microscopes, labels and imaging conditions. Here, we exploit the stereotypic arrangement of proteins in the nuclear pore complex as in situ reference structures to characterize the performance of a variety of microscopy modalities. We created four genome edited cell lines in which we endogenously labeled the nucleoporin Nup96 with mEGFP, SNAP-tag, HaloTag or the photoconvertible fluorescent protein mMaple. We demonstrate their use (1) as three-dimensional resolution standards for calibration and quality control, (2) to quantify absolute labeling efficiencies and (3) as precise reference standards for molecular counting. These cell lines will enable the broader community to assess the quality of their microscopes and labels, and to perform quantitative, absolute measurements.

Adequate nutrition in prisons should constantly be monitored due to the limited possibilities of external control as well as the low catering budget for prison meals and poorly defined requirements in this regard. The aim of the study was to assess the nutritional value of meals served in Polish prisons. Using a computer program, 14-day regular and bland diets from 30 prisons were analyzed. The energy value of the meals and the percentage of energy provided by protein, fat, and carbohydrate contained therein were found to meet the recommendations of the Polish National Food and Nutrition Institute. The amount of minerals supplied with the diet did not cover the recommended dietary allowance (RDA) in the case of calcium and magnesium. Particularly disturbing was the excessive supply of sodium in the regular and bland diets, which covered 537% and 311% of the dietary reference intake (DRI), respectively, as well as phosphorus (194 and 192% of RDA). The largest vitamin deficiencies were recorded for vitamins D and C and folate. An especially excessive supply was observed for vitamins A and B12. The type of diet significantly differentiated the average content of over half of the analyzed components, whereas the season of the year turned out to be statistically insignificant. The results of the present investigations indicate a need for development of more accurate legal provisions to regulate the nutrition in Polish prisons in terms of not only the energy value and macronutrient supply but also the intake of minerals and vitamins.

Lack of data provenance negatively impacts scientific reproducibility and the reliability of genomic data. The ATCC Genome Portal (https://genomes.atcc.org) addresses this by providing data provenance information for microbial whole-genome assemblies originating from authenticated biological materials. To date, we have sequenced 1,579 complete genomes, including 466 type strains and 1,156 novel genomes.

National reference standards (NRSs) for biologics are established through potency estimation by a multicenter joint study of standard materials used in the approval process for national lot release and quality control of vaccines, blood products, and other biologics. In this study, a stability evaluation was conducted to determine whether the potency of NRSs for six blood products was being maintained at a consistent level in Korea. The present study conducted real-time stability tests via in-vivo/in-vitro bioassay on NRSs for blood coagulation factor VIII concentrate (2nd standard), antithrombin concentrate, prekallikrein activator, anti-hepatitis B immunoglobulin, blood coagulation factor IX concentrate, and anti-tetanus human immunoglobulin, as well as a trend analysis using cumulative annual results. The real-time stability test results showed that the mean potency of six NRSs was all within the control limit. In the trend analysis, the potency of NRS for blood coagulation factor VIII concentrate (2nd standard) showed a decreasing trend, while the potency of all other products had been stably maintained. The present study confirmed that the mean potency of NRSs for six blood products had been stably maintained in Korea. The findings of the present study establish a foundation that can ensure the quality of NRSs for biologics in Korea, and it is expected to make a major contribution to the supply of high-quality biologics.

The complexity of microbial communities, combined with technical biases in next-generation sequencing, pose a challenge to metagenomic analysis. Here, we develop a set of internal DNA standards, termed "sequins" (sequencing spike-ins), that together constitute a synthetic community of artificial microbial genomes. Sequins are added to environmental DNA samples prior to library preparation, and undergo concurrent sequencing with the accompanying sample. We validate the performance of sequins by comparison to mock microbial communities, and demonstrate their use in the analysis of real metagenome samples. We show how sequins can be used to measure fold change differences in the size and structure of accompanying microbial communities, and perform quantitative normalization between samples. We further illustrate how sequins can be used to benchmark and optimize new methods, including nanopore long-read sequencing technology. We provide metagenome sequins, along with associated data sets, protocols, and an accompanying software toolkit, as reference standards to aid in metagenomic studies.

National reference standards for anti-HIV-1 antibody are needed to evaluate the performance and maintain the quality control of anti-HIV-1 antibody assays. The aim of this study was to prepare a mixed-titer performance panel and assess its suitability as a national reference standard for anti-HIV-1 antibody according to stability, collaboration, and other studies.

NIST standard reference material (SRM) 2373 was developed to improve the measurements of the HER2 gene amplification in DNA samples. SRM 2373 consists of genomic DNA extracted from five breast cancer cell lines with different amounts of amplification of the HER2 gene. The five components are derived from the human cell lines SK-BR-3, MDA-MB-231, MDA-MB-361, MDA-MB-453, and BT-474. The certified values are the ratios of the HER2 gene copy numbers to the copy numbers of selected reference genes DCK, EIF5B, RPS27A, and PMM1. The ratios were measured using quantitative polymerase chain reaction and digital PCR, methods that gave similar ratios. The five components of SRM 2373 have certified HER2 amplification ratios that range from 1.3 to 17.7. The stability and homogeneity of the reference materials were shown by repeated measurements over a period of several years. SRM 2373 is a well characterized genomic DNA reference material that can be used to improve the confidence of the measurements of HER2 gene copy number.

The estimation of nuclear DNA content has been by far the most popular application of flow cytometry in plants. Because flow cytometry measures relative fluorescence intensities of nuclei stained by a DNA fluorochrome, ploidy determination, and estimation of the nuclear DNA content in absolute units both require comparison to a reference standard of known DNA content. This implies that the quality of the results obtained depends on the standard selection and use. Internal standardization, when the nuclei of an unknown sample and the reference standard are isolated, stained, and measured simultaneously, is mandatory for precise measurements. As DNA peaks representing G1 /G0 nuclei of the sample and standard appear on the same histogram of fluorescence intensity, the quotient of their position on the fluorescence intensity axis provides the quotient of DNA amounts. For the estimation of DNA amounts in absolute units, a number of well-established standards are now available to cover the range of known plant genome sizes. Since there are different standards in use, the standard and the genome size assigned to it has always to be reported. When none of the established standards fits, the introduction of a new standard species is needed. For this purpose, the regression line approach or simultaneous analysis of the candidate standard with several established standards should be prioritized. Moreover, the newly selected standard organism has to fulfill a number of requirements: it should be easy to identify and maintain, taxonomically unambiguous, globally available, with known genome size stability, lacking problematic metabolites, suitable for isolation of sufficient amounts of nuclei, and enabling measurements with low coefficients of variation of DNA peaks, hence suitable for the preparation of high quality samples.

Fingerprint analysis and simultaneous multi-components determination are crucial for the holistic quality control of traditional Chinese medicines (TCMs). Yet, reference standards (RS) are often commercially unavailable and with other shortages, which severely impede the application of these technologies.

The gluten-free diet (GFD) requires special attention from nutritionists due to the potential risk of nutrient deficiencies in its users. This risk may be greater when this type of nutrition is implemented in prisons due to the limited possibilities of external control, a low catering budget for meals, and insufficiently defined recommendations regulating nutrition for prisoners. The aim of the present study was to assess the nutritional value of GFD and regular diet meals served in some Polish prisons and to compare the values to the dietary reference intake (DRI) standards. Using a specialized computer program, 7-day menus of both types of diet provided in 10 prisons were analyzed. The percentage coverage of the DRI was calculated based on the recommendations of the Polish National Food and Nutrition Institute. GFD was characterized by lower average contents of energy and 11 out of 14 essential nutrients, i.e., protein, carbohydrates, dietary fiber, starch, ash, sodium, calcium, iron, zinc, folate, and vitamin B12. The average content of phosphorus, niacin, and riboflavin in the gluten-free diet was higher than that in the regular diet. It was shown that the meals in GFD and the regular diet did not provide the recommended amounts of calcium (38 and 44% DRI, respectively), vitamin D (29 and 30% DRI), vitamin C (86 and 76% DRI), and folate (51 and 56% DRI). In turn, the supply of sodium, phosphorus, copper, and vitamins A and B6 substantially exceeded the recommended levels. The results indicate a need for greater quality control of GFD meals served in catering facilities. It is also necessary to develop legal provisions that will regulate more specifically the nutrition for prisoners in terms of an adequate supply of minerals and vitamins.

BACKGROUND Chagas disease, resulting from Trypanosoma cruzi infections, continues to be a health concern mainly in Latin American countries where the parasite is endemic. The laboratory diagnosis of a chronic infection is determined through serological assays for antibodies against T. cruzi and several tests are available that differ in key components, formats and methodologies. To date, no single test meets the criteria of a gold standard. The situation is further complicated by the difficulties associated with performance comparisons between different immunoassays or methodologies executed at different times and geographical areas. OBJECTIVE To improve the diagnosis of Chagas disease, the WHO coordinated the development of two International Biological Reference Standards for antibodies against anti-T. cruzi: NIBSC 09/186 and NIBSC 09/188 that respectively represent geographical regions with the highest prevalence of TcII and TcI lineages of the parasite. METHODS The principle goal of this study was to verify the behavior of these standards when assayed by several commercially available serological tests that employ different methods to capture and detect human anti-T. cruzi antibodies. FINDINGS AND MAIN CONCLUSIONS The results reinforce the recommendation that these standards be considered for performance evaluations of commercialised immunoassays and should be an integral step in the development of new test components or assay paradigms.

The outbreak of human infections of a novel avian influenza virus A (H7N9) prompted the development of the vaccines against this virus. Like all types of influenza vaccines, H7N9 vaccine must be tested for its potency prior to being used in humans. However, the unavailability of international reference reagents for the potency determination of H7N9 vaccines substantially hinders the progress in vaccine development. To facilitate clinical development, we enlisted 5 participants in a collaborative study to develop critical reagents used in Single Radial Immunodiffusion (SRID), the currently acceptable assay for potency determination of influenza vaccine. Specifically, the hemagglutinin (HA) content of one vaccine bulk for influenza A (H7N9), herein designated as Primary Liquid Standard (PLS), was determined by SDS-PAGE. In addition, the freeze-dried antigen references derived from PLS were prepared to enhance the stability for long term storage. The final HA content of lyophilized antigen references were calibrated against PLS by SRID assay in a collaborative study. Importantly, application of these national reference standards to potency analyses greatly facilitated the development of H7N9 vaccines in China.

Eukaryotic genomes contain many nongenic elements that function in gene regulation, chromosome organization, recombination, repair, or replication, and mutation of those elements can affect genome function and cause disease. Although numerous epigenomic studies provide high coverage of gene regulatory regions, those data are not usually exposed in traditional genome annotation and can be difficult to access and interpret without field-specific expertise. The National Center for Biotechnology Information (NCBI) therefore provides RefSeq Functional Elements (RefSeqFEs), which represent experimentally validated human and mouse nongenic elements derived from the literature. The curated data set is comprised of richly annotated sequence records, descriptive records in the NCBI Gene database, reference genome feature annotation, and activity-based interactions between nongenic regions, target genes, and each other. The data set provides succinct functional details and transparent experimental evidence, leverages data from multiple experimental sources, is readily accessible and adaptable, and uses a flexible data model. The data have multiple uses for basic functional discovery, bioinformatics studies, genetic variant interpretation; as known positive controls for epigenomic data evaluation; and as reference standards for functional interactions. Comparisons to other gene regulatory data sets show that the RefSeqFE data set includes a wider range of feature types representing more areas of biology, but it is comparatively smaller and subject to data selection biases. RefSeqFEs thus provide an alternative and complementary resource for experimentally assayed functional elements, with future data set growth expected.

Clinical trials using recombinant adeno-associated virus (rAAV) vectors have demonstrated efficacy and a good safety profile. Although the field is advancing quickly, vector analytics and harmonization of dosage units are still a limitation for commercialization. AAV reference standard materials (RSMs) can help ensure product safety by controlling the consistency of assays used to characterize rAAV stocks. The most widely utilized unit of vector dosing is based on the encapsidated vector genome. Quantitative polymerase chain reaction (qPCR) is now the most common method to titer vector genomes (vg); however, significant inter- and intralaboratory variations have been documented using this technique. Here, RSMs and rAAV stocks were titered on the basis of an inverted terminal repeats (ITRs) sequence-specific qPCR and we found an artificial increase in vg titers using a widely utilized approach. The PCR error was introduced by using single-cut linearized plasmid as the standard curve. This bias was eliminated using plasmid standards linearized just outside the ITR region on each end to facilitate the melting of the palindromic ITR sequences during PCR. This new "Free-ITR" qPCR delivers vg titers that are consistent with titers obtained with transgene-specific qPCR and could be used to normalize in-house product-specific AAV vector standards and controls to the rAAV RSMs. The free-ITR method, including well-characterized controls, will help to calibrate doses to compare preclinical and clinical data in the field.

NASA has made great strides in the past five years to develop a suite of instruments for the International Space Station in order to perform molecular biology in space. However, a key piece of equipment that has been lacking is an instrument that can extract nucleic acids from an array of complex human and environmental samples. The Omics in Space team has developed the μTitan (simulated micro(μ) gravity tested instrument for automated nucleic acid) system capable of automated, streamlined, nucleic acid extraction that is adapted for use under microgravity. The μTitan system was validated using a whole cell microbial reference (WCMR) standard comprised of a suspension of nine bacterial strains, titrated to concentrations that would challenge the performance of the instrument, as well as to determine the detection limits for isolating DNA. Quantitative assessment of system performance was measured by comparing instrument input challenge dose vs recovery by Qubit spectrofluorometry, qPCR, Bioanalyzer, and Next Generation Sequencing. Overall, results indicate that the μTitan system performs equal to or greater than a similar commercially available, earth-based, automated nucleic acid extraction device. The μTitan system was also tested in Yellowstone National Park (YNP) with the WCMR, to mimic a remote setting, with limited resources. The performance of the device at YNP was comparable to that in a laboratory setting. Such a portable, field-deployable, nucleic extraction system will be valuable for environmental microbiology, as well as in health care diagnostics.

Quantitative nuclear magnetic resonance (qNMR) spectroscopy is employed by an increasing number of analytical and industrial laboratories for the assignment of content and quantitative determination of impurities. Within the last few years, it was demonstrated that (1)H qNMR can be performed with high accuracy leading to measurement uncertainties below 1 % relative. It was even demonstrated that the combination of (1)H qNMR with metrological weighing can lead to measurement uncertainties below 0.1 % when highly pure substances are used. Although qNMR reference standards are already available as certified reference materials (CRM) providing traceability on the basis of (1)H qNMR experiments, there is an increasing demand for purity assays on phosphorylated organic compounds and metabolites requiring CRM for quantification by (31)P qNMR. Unfortunately, the number of available primary phosphorus standards is limited to a few inorganic CRM which only can be used for the analysis of water-soluble analytes but fail when organic solvents must be employed. This paper presents the concept of value assignment by (31)P qNMR measurements for the development of CRM and describes different approaches to establish traceability to primary Standard Reference Material from the National Institute of Standards and Technology (NIST SRM). Phosphonoacetic acid is analyzed as a water-soluble CRM candidate, whereas triphenyl phosphate is a good candidate for the use as qNMR reference material in organic solvents. These substances contain both nuclei, (1)H and (31)P, and the concept is to show that it is possible to indirectly quantify a potential phosphorus standard via its protons using (1)H qNMR. The same standard with its assigned purity can then be used for the quantification of an analyte via its phosphorus using (31)P qNMR. For the validation of the concept, triphenyl phosphate and phosphonoacetic acid have been used as (31)P qNMR standards to determine the purity of the analyte tris(2-chloroethyl) phosphate, and the resulting purity values perfectly overlap within their expanded measurement uncertainties.

Lentiviral vectors (LV) have emerged as a robust technology for therapeutic gene delivery into human cells as advanced medicinal products. As these products are increasingly commercialized, there are concomitant demands for their characterization to ensure safety, efficacy and consistency. Standards are essential for accurately measuring parameters for such product characterization. A critical parameter is the vector copy number (VCN) which measures the genetic dose of a transgene present in gene-modified cells. Here we describe a set of clonal Jurkat cell lines with defined copy numbers of a reference lentiviral vector integrated into their genomes. Genomic DNA was characterized for copy number, genomic integrity and integration coordinates and showed uniform performance across independent quantitative PCR assays. Stability studies during continuous long-term culture demonstrated sustained renewability of the reference standard source material. DNA from the Jurkat VCN standards would be useful for control of quantitative PCR assays for VCN determination in LV gene-modified cellular products and clinical samples.