SEAOP: a statistical ensemble approach for outlier detection in quantitative proteomics data.

Quality control in quantitative proteomics is a persistent challenge, particularly in identifying and managing outliers. Unsupervised learning models, which rely on data structure rather than predefined labels, offer potential solutions. However, without clear labels, their effectiveness might be compromised. Single models are susceptible to the randomness of parameters and initialization, which can result in a high rate of false positives. Ensemble models, on the other hand, have shown capabilities in effectively mitigating the impacts of such randomness and assisting in accurately detecting true outliers. Therefore, we introduced SEAOP, a Python toolbox that utilizes an ensemble mechanism by integrating multi-round data management and a statistics-based decision pipeline with multiple models. Specifically, SEAOP uses multi-round resampling to create diverse sub-data spaces and employs outlier detection methods to identify candidate outliers in each space. Candidates are then aggregated as confirmed outliers via a chi-square test, adhering to a 95% confidence level, to ensure the precision of the unsupervised approaches. Additionally, SEAOP introduces a visualization strategy, specifically designed to intuitively and effectively display the distribution of both outlier and non-outlier samples. Optimal hyperparameter models of SEAOP for outlier detection were identified by using a gradient-simulated standard dataset and Mann-Kendall trend test. The performance of the SEAOP toolbox was evaluated using three experimental datasets, confirming its reliability and accuracy in handling quantitative proteomics.

Development of a nine-variant reference material panel to standardize cell-free DNA detection.

Detection of specific gene mutations in cell-free DNA (cfDNA) serves as a valuable cancer biomarker and is increasingly being explored as an appealing alternative to tissue-based methods. However, the lack of available reference materials poses challenges in accurately evaluating the performance of different assays. In this study, we present the development of a comprehensive reference material panel for cfDNA detection, encompassing nine hotspot mutations in KRAS/BRAF/EGFR/PIK3CA at three variant allele frequencies (VAFs), ranging from 0.33 to 23.9%. To mimic cfDNA, these reference materials were generated by enzymatically digesting cell-line DNA into approximately 154-bp to 173-bp fragments using a laboratory-developed reaction system. The VAFs for each variation were precisely determined through validated digital PCR assays with high accuracy. Furthermore, the reliability and applicability of this panel were confirmed through two independent NGS assays, yielding concordant results. Collectively, our findings suggest that this novel reference material panel holds great potential for validation, evaluation, and quality control processes associated with liquid biopsy assays.

Reliable biological and multi-omics research through biometrology.

Metrology is the science of measurement and its applications, whereas biometrology is the science of biological measurement and its applications. Biometrology aims to achieve accuracy and consistency of biological measurements by focusing on the development of metrological traceability, biological reference measurement procedures, and reference materials. Irreproducibility of biological and multi-omics research results from different laboratories, platforms, and analysis methods is hampering the translation of research into clinical uses and can often be attributed to the lack of biologists' attention to the general principles of metrology. In this paper, the progresses of biometrology including metrology on nucleic acid, protein, and cell measurements and its impacts on the improvement of reliability and comparability in biological research are reviewed. Challenges in obtaining more reliable biological and multi-omics measurements due to the lack of primary reference measurement procedures and new standards for biological reference materials faced by biometrology are discussed. In the future, in addition to establishing reliable reference measurement procedures, developing reference materials from single or multiple parameters to multi-omics scale should be emphasized. Thinking in way of biometrology is warranted for facilitating the translation of high-throughput omics research into clinical practices.

Integrated Microwell Array-Based Microfluidic Chip with a Hand-Held Smartphone-Controlled Device for Nucleic Acid Detection.

In this study, we designed a highly integrated microfluidic chip for nucleic acid extraction, amplification, and detection. Magnetic beads, which are used to capture nucleic acids on the chip, are trapped in the microwell arrays in a one-well-one-bead manner after local surface modification of the inner faces of the microwells. On-chip liquid introduction, delivery, and mixing are all carried out manually with one syringe and no other equipment. A hand-held device with precise temperature control and high-quality imaging is developed, which is only 2.3 cubic decimeters in volume and 1.2 kg in weight. Via the use of the Internet for wireless communication, the experiment and data analysis after inserting the chip into the device can be conducted by a smartphone anywhere there is an Internet connection. We carried out reverse transcription loop-mediated isothermal amplification (RT-LAMP) on the chip with the hand-held device. SARS-CoV-2 pseudoviruses are extracted, reverse transcribed, amplified, and detected on the chip with the hand-held device with satisfactory results. Thus, a highly integrated, easy-to-operate, and rapid nucleic acid detection microfluidic chip with a hand-held smartphone-controlled device is proposed, and this new platform for nucleic acid detection shows great potential for mobile point-of-care testing (POCT).

Reference material development for detection of human respiratory syncytial virus using digital PCR.

Nucleic acid testing is a powerful tool for the detection of various pathogens. Respiratory syncytial virus (RSV) is a major cause of acute respiratory infection, especially in young children and infants. To improve the confidence and reliability of nucleic acid testing results for RSV, reference materials (RMs) of both type A and B of RSV were developed by the National Institute of Metrology, China, code numbers NIM-RM 4057 and 4058. The reference material was composed of in vitro transcribed RNA containing the nucleocapsid (N) gene, matrix (M) gene, and partial polymerase (L) gene of RSV. A duplex reverse transcription digital PCR method was established with limit of blank (LoB), limit of detection (LoD) and limit of quantification (LoQ) of 2, 5, and 23 copies per reaction for RSV-A and 4, 8, and 20 copies per reaction for RSV-B. The certified value and expanded uncertainty (U, k = 2) of the two RMs were determined to be (6.1 ± 1.4) × 104 copies/µL for RSV-A and (5.3 ± 1.2) × 104 copies/µL for RSV-B. The developed RMs can be used as standards to evaluate the performance of RSV detection assays.

Establishment and evaluation of digital PCR methods for HER2 copy number variation in breast cancer.

Accurate measurement of human epidermal growth factor receptor 2 (HER2) copy number variation (CNV) is very important for guiding the tumor target therapy in breast cancer. Digital PCR (dPCR) is a sensitive and an absolute quantitative method, which can be used to detect HER2 CNV. Three HER2 exon-specific digital PCR assays along with three new reference genes assays (homo sapiens ribonuclease P RNA component H1 (RPPH1), glucose-6-phosphate isomerase (GPI), and chromosome 1 open reading frame 43 (C1ORF43), on different chromosomes) were established and validated by using standard reference material, 8 different cell lines and 110 clinical Formalin-fixed and paraffin-embedded (FFPE) samples. DPCR can achieve precise quantification of HER2 CNV by calculating the ratio of HER2/reference gene. The positive and negative coincidence rates were 98% (53/54) and 95% (53/56), respectively, compared with fluorescence in situ hybridization (FISH) diagnostic result 110 of FFPE samples. The common reference gene CEP17 used for FISH diagnostic was not suitable as single reference gene for HER2 CNV measurements by dPCR. The best practice of HER2 CNV determination by dPCR is to conduct the three duplex assays of H1 (HER2 exon 4) with the proposed three new reference genes, with a positive cut-off value of H1/RPPH1 ≥ 2.0 or H1/averaged reference gene ≥ 2.0. The proposed dPCR method in our study can accurately provide absolute copy number of HER2 and reference gene on an alternative chromosome, thus avoiding false negative caused by polysomy of chromosome 17. The improved molecular typing and diagnosis of breast cancer will better guide clinical medication.

Development of highly accurate digital PCR method and reference material for monkeypox virus detection.

Human monkeypox has attracted attention recently. Monkeypox virus (MPXV) keeps evolving as it spreading around the world rapidly, which may threaten the health of more and more people. Here, we have developed a high order reference method based on digital PCR (dPCR) for MPXV detection, of which the limits of quantification (LoQ) and detection (LoD) are 38 and 6 copies/reaction, respectively. Pseudovirus reference materials (RM) containing the conserved F3L gene has been developed, and the homogeneity assessment showed that the RM was homogeneous. The reference value with its expanded uncertainty determined by the established dPCR is (2.74 ± 0.46) × 103 copies/µL. Six different MPXV test kits were accessed by the RM. Four out of six test kits cannot reach their claimed LoDs. The poor analytical sensitivity might cause false-negative results, which lead to incorrect diagnosis and treatment. The establishment of a high order reference method of dPCR and pseudovirus RM is very useful for improving the accuracy and reliability of MPXV detection.

A culture-free method for rapidly and accurately quantifying active SARS-CoV-2.

Determining the quantity of active virus is the most important basis to judge the risk of virus infection, which usually relies on the virus median tissue culture infectious dose (TCID50) assay performed in a biosafety level 3 laboratory within 5-7 days. We have developed a culture-free method for rapid and accurate quantification of active severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by targeting subgenomic RNA (sgRNA) based on reverse transcription digital PCR (RT-dPCR). The dynamic range of quantitative assays for sgRNA-N and sgRNA-E by RT-dPCR was investigated, and the result showed that the limits of detection (LoD) and quantification (LoQ) were 2 copies/reaction and 10 copies/reaction, respectively. The delta strain (NMDC60042793) of SARS-CoV-2 was cultured at an average titer of 106.13 TCID50/mL and used to evaluate the developed quantification method. Copy number concentrations of the cultured SARS-CoV-2 sgRNA and genomic RNA (gRNA) gave excellent linearity (R2 = 0.9999) with SARS-CoV-2 titers in the range from 500 to 105 TCID50/mL. Validation of 63 positive clinical samples further proves that the quantification of sgRNA-N by RT-dPCR is more sensitive for active virus quantitative detection. It is notable that we can infer the active virus titer through quantification of SARS-CoV-2 sgRNA based on the linear relationship in a biosafety level 2 laboratory within 3 h. It can be used to timely and effectively identify infectious patients and reduce unnecessary isolation especially when a large number of COVID-19 infected people impose a burden on medical resources.

Establishment of Digital PCR Method and Reference Material for Adenoviruses 40 and 41.

Coinfection with human adenovirus (HAdV) and SARS-CoV-2 has been associated with acute hepatitis in children with unknown etiology. Similar cases have been reported in many countries, and HAdV 40 and HAdV 41 have been identified. The quantification method is established based on digital PCR (dPCR) for HAdV 40/41, which is more convenient for low-concentration virus detection. The limit of detections of HAdV 40/41 dPCR were 4 and 5 copies/µL. Pseudovirus reference material (RM) that contains the highly conserved HEXON gene was developed and quantified with the dPCR method. The assigned values with expanded uncertainty were (1.43 ± 0.35) × 103 copies/µL for HAdV 40 RM and (1.21 ± 0.28) × 103 copies/µL for HAdV 41 RM. The values could be reproduced on multiple platforms. The dPCR method and pseudovirus RMs contribute to the improved accuracy of HAdV 40/41 detection, which is crucial for clinical diagnosis.

International co-validation on absolute quantification of single nucleotide variants of KRAS by digital PCR.

The precise quantification of KRAS single nucleotide variant (SNV) is critical for the treatment and prognosis of lung and colorectal cancer. Validation of digital PCR (dPCR) as a method for accurate quantification of KRAS SNV has great clinical importance. An international co-validation on absolute quantification of KRAS SNV by dPCR was conducted among three national measurement institutes (NMIs) from China (NIM), South Korea (KRISS), and Japan (NMIJ). A candidate reference material (RM) was provided by NIM and three measurands were reported: copy number concentration (Tc) of KRAS G12A mutation and wild type and KRAS G12A fractional abundance (FA). Homogeneity and stability assessment showed that the study materials provided by NIM were sufficiently homogeneous and stable during the study period. En number performance statistics was used to evaluate equivalence of the study among the three NMIs. All En values for both Tc and KRAS G12A FA≤1 showed good agreement and consistency with the reference value within the expanded uncertainty. This indicates that dPCR with full uncertainty evaluation can serve as a candidate primary reference measurement procedure (PRMP) for the KRAS SNV measurement and value assignment of reference materials.

Accurate quantification of SARS-CoV-2 RNA by isotope dilution mass spectrometry and providing a correction of reverse transcription efficiency in droplet digital PCR.

The novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 505 million confirmed cases, including over 6 million deaths. Reference materials (RMs) of SARS-CoV-2 RNA played a crucial role in performance evaluation and quality control of testing laboratories. As the potential primary characterization method of RMs, reverse transcription digital PCR (RT-dPCR) measures the copy number of RNA, but the accuracy of reverse transcription (RT) efficiency has yet to be confirmed. This study established a method of enzymatic digestion followed by isotope dilution mass spectrometry (IDMS), which does not require an RT reaction, to quantify in vitro-transcribed SARS-CoV-2 RNA. RNA was digested to nucleotide monophosphate (NMP) within 15 min and analyzed by IDMS within 5 min. The consistency among the results of four different NMPs demonstrated the reliability of the proposed method. Compared to IDMS, the quantitative result of RT-dPCR turned out to be about 10% lower, possibly attributed to the incompleteness of the reverse transcription process. Therefore, the proposed approach could be valuable and reliable for quantifying RNA molecules and evaluating the RT efficiency of RT-based methods.

An efficient method to identify virus-specific TCRs for TCR-T cell immunotherapy against virus-associated malignancies.

Adoptive transfer of T cells genetically engineered with a T cell receptor (TCR) is a promising cancer treatment modality that requires the identification of TCRs with good characteristics. Most T cell cloning methods involve a stringent singularization process, which necessitates either tedious hands-on operations or high cost. We present an efficient and nonstringent cloning approach based on existing techniques. We hypothesize that after elimination of most nonspecific T cells, a clonotype with high quality could outcompete other clonotypes and finally form a predominant population. This TCR identification method can be used to clone virus-specific TCRs efficiently from cancer patients and is easily adoptable by any laboratory.

Interlaboratory assessment of quantification of SARS-CoV-2 RNA by reverse transcription digital PCR.

The pandemic of the novel coronavirus disease 2019 (COVID-19) has caused severe harm to the health of people all around the world. Molecular detection of the pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), played a crucial role in the control of the disease. Reverse transcription digital PCR (RT-dPCR) has been developed and used in the detection of SARS-CoV-2 RNA as an absolute quantification method. Here, an interlaboratory assessment of quantification of SARS-CoV-2 RNA was organized by the National Institute of Metrology, China (NIMC), using in vitro transcribed RNA samples, among ten laboratories on six different dPCR platforms. Copy number concentrations of three genes of SARS-CoV-2 were measured by all participants. Consistent results were obtained with dispersion within 2.2-fold and CV% below 23% among different dPCR platforms and laboratories, and Z' scores of all the reported results being satisfactory. Possible reasons for the dispersion included PCR assays, partition volume, and reverse transcription conditions. This study demonstrated the comparability and applicability of RT-dPCR method for quantification of SARS-CoV-2 RNA and showed the capability of the participating laboratories at SARS-CoV-2 test by RT-dPCR platform.

Fluorometric determination of HIV DNA using molybdenum disulfide nanosheets and exonuclease III-assisted amplification.

A convenient and ultrasensitive fluorometric method is described for the determination of HIV DNA. It exploits the strong difference in the affinities of MoS2 nanosheets for long ssDNA versus short oligonucleotide fragments. In addition, efficient signal amplification is accomplished by exonuclease III-assisted target recycling. When absorbed on the MoS2 nanosheets, the fluorescence of the FAM-labeled ssDNA probe (FP) is quenched. However, in the presence of HIV DNA, the FP hybridizes with target to form a duplex. As a result, the FP in the duplex will be stepwise hydrolyzed into short fragments by Exo III, and the fluorescence signal thus is retained because short fragments have low affinity for the MoS2 nanosheets. By using the Exo III-assisted target recycling amplification, the detection sensitivity is strongly improved. The sensor can detect DNA in a concentration as low as 5.3 pM (at an S/N ratio of 3), and the analytical range extends from 0.01 nM to 10 nM. The assay is simple, sensitive and specific, and conceivably represents a valuable tool in clinical studies related to the HIV. Graphical abstract Schematic presentation of fluorometric determination of HIV DNA based on molybdenum disulfide nanosheets and Exo III. When the fluorescence-tagged ssDNA probe hybridized with target to form a duplex, the Exo III-assisted target recycling amplification is generated. The method can detect as low as 5.3 pM HIV DNA.

Genome-wide analysis of long non-coding RNAs and their role in postnatal porcine testis development.

A comprehensive and systematic understanding of the roles of lncRNAs in the postnatal development of the pig testis has still not been achieved. In the present study, we obtained more than one billion clean reads and identified 15,528 lncRNA transcripts; these transcripts included 5032 known and 10,496 novel porcine lncRNA transcripts and corresponded to 10,041 lncRNA genes. Pairwise comparisons identified 449 known and 324 novel lncRNAs that showed differential expression patterns. GO and KEGG pathway enrichment analyses revealed that the targeted genes were involved in metabolic pathways regulating testis development and spermatogenesis, such as the TGF-beta pathway, the PI3K-Akt pathway, the Wnt/ß-catenin pathway, and the AMPK pathway. Using this information, we predicted some lncRNAs and coding gene pairs were predicted that may function in testis development and spermatogenesis; these are listed in detail. This study has provided the most comprehensive catalog to date of lncRNAs in the postnatal pig testis and will aid our understanding of their functional roles in testis development and spermatogenesis.

International Comparison of Enumeration-Based Quantification of DNA Copy-Concentration Using Flow Cytometric Counting and Digital Polymerase Chain Reaction.

Enumeration-based determination of DNA copy-concentration was assessed through an international comparison among national metrology institutes (NMIs) and designated institutes (DIs). Enumeration-based quantification does not require a calibration standard thereby providing a route to "absolute quantification", which offers the potential for reliable value assignments of DNA reference materials, and International System of Units (SI) traceability to copy number 1 through accurate counting. In this study, 2 enumeration-based methods, flow cytometric (FCM) counting and the digital polymerase chain reaction (dPCR), were compared to quantify a solution of the pBR322 plasmid at a concentration of several thousand copies per microliter. In addition, 2 orthogonal chemical-analysis methods based on nucleotide quantification, isotope-dilution mass spectrometry (IDMS) and capillary electrophoresis (CE) were applied to quantify a more concentrated solution of the plasmid. Although 9 dPCR results from 8 laboratories showed some dispersion (relative standard deviation [RSD] = 11.8%), their means were closely aligned with those of the FCM-based counting method and the orthogonal chemical-analysis methods, corrected for gravimetric dilution factors. Using the means of dPCR results, the RSD of all 4 methods was 1.8%, which strongly supported the validity of the recent enumeration approaches. Despite a good overall agreement, the individual dPCR results were not sufficiently covered by the reported measurement uncertainties. These findings suggest that some laboratories may not have considered all factors contributing to the measurement uncertainty of dPCR, and further investigation of this possibility is warranted.

The role of ubiquitin-proteasome pathway in spermatogenesis.

Ubiquitin-proteasome pathway (UPP) is the main pathway of protein degradation in eukaryotic cells. The UPP plays very important roles in cell cycle progression, apoptosis, stress response and growth and development through regulating protein interaction, protein activity, protein localization and signal transduction. Previous studies have shown that the UPP is essential for regulating acrosome and tail biogenesis during spermatogenesis in human and animals. The dysregulation of UPP during spermatogenesis results in sperm deformity and reduced sperm motility and leads to reproductive system diseases such as oligospermatism, infertility and testicular tumors. In this review, we summarized the signal transduction and regulation mechanism of UPP in spermatogenesis, which may provide references for future studies.

Effects of Transgenic Bt+CpTI cotton on the abundance and diversity of rhizosphere ammonia oxidizing bacteria and archaea.

Genetically modified crops (GMCs) hold great promise for improving agricultural output, but at the same time present challenges in terms of environmental safety assessment. Ammonia oxidizers, including ammonia oxidizing bacteria (AOB) and archaea (AOA), are very important functional microbial groups in nitrogen cycle. The abundance and diversity of AOA and AOB in the rhizosphere of genetically modified cotton (SGK321) and non-GM cotton (SY321) across growth stages were investigated using real time quantitative PCR (qPCR) and terminal restriction fragment length polymorphism (T-RFLP). Results showed that cotton genotype had a significant effect on the change in abundance of AOA and AOB, as indicated by amoA copy number. Variations in AOB abundance in rhizosphere of SY321 differed from those in SGK321. The number of AOB in the rhizosphere of SY321 fluctuated considerably: It dramatically decreased from 1.2?106 copies g-1 dry soil to 3?105 copies g-1 dry soil during the flowering stage and then increased to 1.1?106 copies g-1 and 1.5?106 copies g-1 at the belling and boll opening stages, respectively. However, abundance of AOB in the rhizosphere of SGK321 was relatively stable during all the stages of growth. The effect of SGK321 and SY321 on AOA number was quite similar to that of AOB: AOA abundance in SGK321 increased smoothly from 1.0 ?105 copies g-1 dry soil to 1.4?106 copies g-1 dry soil during growth, but that in SY321 fluctuated. Correspondence analysis (CA), canonical CA (CCA), and partial CCA (pCCA) of T-RFLP profiles of AOA and AOB showed that AOB community changed across growth stages in both cotton genotypes, and cotton genotype was the most important factor affecting the AOA community. In conclusion, the current findings indicated no adverse effect of GM cotton on functional microorganisms.

[Effects of planting transgenic Bt + CpTI cotton on rhizosphere denitrifier abundance and diversity].

OBJECTIVE: To evaluate the effect of planting genetically modified cotton on soil denitrifer. METHODS: The impact of transgenic Bt + CpTI cotton (SGK321) and its receptor cotton (SY321) on rhizosphere denitrifier abundance and diversity were investigated by using quantitative PCR (qPCR) and terminal restriction fragment length polymorphism (T-RFLP). We collected rhizosphere soil before cotton planting (Pre) and along with the cotton growth stage (budding, flowering, belling and boll opening). RESULTS: The abundance of denitrifier in both cottons changed significantly across the growth stage, but the variation tendency was different. In the rhizosphere of transgenic cotton, the denitrifier abundance increased from 3.12 x 10(6) copies/g dry soil (Pre) to 2.81 x 10(7) copies/g dry soil (belling). The denitrifier abundance in non-transgenic cotton was significantly affected by the growth stage: increased at budding, decreased at flowering, and then increased at belling. Canonical correspondence analysis and partial canonical correspondence analysis show that the denitrifier diversity was more correlated with pH, concentration of NO3- and budding and flowering. Additionally, cotton genotype was an important factor of influencing the diversity of denitrifier. CONCLUSIONS: This indicates the abundance and diversity were influenced by both the cotton growth stage and the cotton genotype by adjusting the soil pH and concentration of NO3-. Planting of transgenic Bt + CpTI cotton leads an increase in the soil pH, which results in an increase in abundance and diversity of denitrifier.

Evaluation of droplet digital PCR for characterizing plasmid reference material used for quantifying ammonia oxidizers and denitrifiers.

DNA reference materials of certified value have a critical function in many analytical processes of DNA measurement. Quantification of amoA genes in ammonia oxidizing bacteria (AOB) and archaea (AOA), and of nirS and nosZ genes in the denitrifiers is very important for determining their distribution and abundance in the natural environment. A plasmid reference material containing nirS, nosZ, amoA-AOB, and amoA-AOA is developed to provide a DNA standard with copy number concentration for ensuring comparability and reliability of quantification of these genes. Droplet digital PCR (ddPCR) was evaluated for characterization of the plasmid reference material. The result revealed that restriction endonuclease digestion of plasmids can improve amplification efficiency and minimize the measurement bias of ddPCR. Compared with the conformation of the plasmid, the size of the DNA fragment containing the target sequence and the location of the restriction site relative to the target sequence are not significant factors affecting plasmid quantification by ddPCR. Liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) was used to provide independent data for quantifying the plasmid reference material. The copy number concentration of the digested plasmid determined by ddPCR agreed well with that determined by LC-IDMS, improving both the accuracy and reliability of the plasmid reference material. The reference value, with its expanded uncertainty (k = 2), of the plasmid reference material was determined to be (5.19 ± 0.41) × 10(9) copies µL(-1) by averaging the results of two independent measurements. Consideration of the factors revealed in this study can improve the reliability and accuracy of ddPCR; thus, this method has the potential to accurately quantify DNA reference materials.