Background The Infinium EPIC array measures the methylation status of > 850,000 CpG sites. The EPIC BeadChip uses a two-array design: Infinium Type I and Type II probes. These probe types exhibit different technical characteristics which may confound analyses. Numerous normalization and pre-processing methods have been developed to reduce probe type bias as well as other issues such as background and dye bias.
Methods This study evaluates the performance of various normalization methods using 16 replicated samples and three metrics: absolute beta-value difference, overlap of non-replicated CpGs between replicate pairs, and effect on beta-value distributions. Additionally, we carried out Pearson’s correlation and intraclass correlation coefficient (ICC) analyses using both raw and SeSAMe 2 normalized data.
Results The method we define as SeSAMe 2, which consists of the application of the regular SeSAMe pipeline with an additional round of QC, pOOBAH masking, was found to be the best-performing normalization method, while quantile-based methods were found to be the worst performing methods. Whole-array Pearson’s correlations were found to be high. However, in agreement with previous studies, a substantial proportion of the probes on the EPIC array showed poor reproducibility (ICC < 0.50). The majority of poor-performing probes have beta values close to either 0 or 1, and relatively low standard deviations. These results suggest that probe reliability is largely the result of limited biological variation rather than technical measurement variation. Importantly, normalizing the data with SeSAMe 2 dramatically improved ICC estimates, with the proportion of probes with ICC values > 0.50 increasing from 45.18% (raw data) to 61.35% (SeSAMe 2). Methods

Study Participants and Samples

The whole blood samples were obtained from the Health, Well-being and Aging (Saúde, Ben-estar e Envelhecimento, SABE) study cohort. SABE is a cohort of census-withdrawn elderly from the city of São Paulo, Brazil, followed up every five years since the year 2000, with DNA first collected in 2010. Samples from 24 elderly adults were collected at two time points for a total of 48 samples. The first time point is the 2010 collection wave, performed from 2010 to 2012, and the second time point was set in 2020 in a COVID-19 monitoring project (9±0.71 years apart). The 24 individuals were 67.41±5.52 years of age (mean ± standard deviation) at time point one; and 76.41±6.17 at time point two and comprised 13 men and 11 women.

All individuals enrolled in the SABE cohort provided written consent, and the ethic protocols were approved by local and national institutional review boards COEP/FSP/USP OF.COEP/23/10, CONEP 2044/2014, CEP HIAE 1263-10, University of Toronto RIS 39685.

Blood Collection and Processing

Genomic DNA was extracted from whole peripheral blood samples collected in EDTA tubes. DNA extraction and purification followed manufacturer’s recommended protocols, using Qiagen AutoPure LS kit with Gentra automated extraction (first time point) or manual extraction (second time point), due to discontinuation of the equipment but using the same commercial reagents. DNA was quantified using Nanodrop spectrometer and diluted to 50ng/uL. To assess the reproducibility of the EPIC array, we also obtained technical replicates for 16 out of the 48 samples, for a total of 64 samples submitted for further analyses. Whole Genome Sequencing data is also available for the samples described above.

Characterization of DNA Methylation using the EPIC array

Approximately 1,000ng of human genomic DNA was used for bisulphite conversion. Methylation status was evaluated using the MethylationEPIC array at The Centre for Applied Genomics (TCAG, Hospital for Sick Children, Toronto, Ontario, Canada), following protocols recommended by Illumina (San Diego, California, USA).

Processing and Analysis of DNA Methylation Data

The R/Bioconductor packages Meffil (version 1.1.0), RnBeads (version 2.6.0), minfi (version 1.34.0) and wateRmelon (version 1.32.0) were used to import, process and perform quality control (QC) analyses on the methylation data. Starting with the 64 samples, we first used Meffil to infer the sex of the 64 samples and compared the inferred sex to reported sex. Utilizing the 59 SNP probes that are available as part of the EPIC array, we calculated concordance between the methylation intensities of the samples and the corresponding genotype calls extracted from their WGS data. We then performed comprehensive sample-level and probe-level QC using the RnBeads QC pipeline. Specifically, we (1) removed probes if their target sequences overlap with a SNP at any base, (2) removed known cross-reactive probes (3) used the iterative Greedycut algorithm to filter out samples and probes, using a detection p-value threshold of 0.01 and (4) removed probes if more than 5% of the samples having a missing value. Since RnBeads does not have a function to perform probe filtering based on bead number, we used the wateRmelon package to extract bead numbers from the IDAT files and calculated the proportion of samples with bead number < 3. Probes with more than 5% of samples having low bead number (< 3) were removed. For the comparison of normalization methods, we also computed detection p-values using out-of-band probes empirical distribution with the pOOBAH() function in the SeSAMe (version 1.14.2) R package, with a p-value threshold of 0.05, and the combine.neg parameter set to TRUE. In the scenario where pOOBAH filtering was carried out, it was done in parallel with the previously mentioned QC steps, and the resulting probes flagged in both analyses were combined and removed from the data.

Normalization Methods Evaluated

The normalization methods compared in this study were implemented using different R/Bioconductor packages and are summarized in Figure 1. All data was read into R workspace as RG Channel Sets using minfi’s read.metharray.exp() function. One sample that was flagged during QC was removed, and further normalization steps were carried out in the remaining set of 63 samples. Prior to all normalizations with minfi, probes that did not pass QC were removed. Noob, SWAN, Quantile, Funnorm and Illumina normalizations were implemented using minfi. BMIQ normalization was implemented with ChAMP (version 2.26.0), using as input Raw data produced by minfi’s preprocessRaw() function. In the combination of Noob with BMIQ (Noob+BMIQ), BMIQ normalization was carried out using as input minfi’s Noob normalized data. Noob normalization was also implemented with SeSAMe, using a nonlinear dye bias correction. For SeSAMe normalization, two scenarios were tested. For both, the inputs were unmasked SigDF Sets converted from minfi’s RG Channel Sets. In the first, which we call “SeSAMe 1”, SeSAMe’s pOOBAH masking was not executed, and the only probes filtered out of the dataset prior to normalization were the ones that did not pass QC in the previous analyses. In the second scenario, which we call “SeSAMe 2”, pOOBAH masking was carried out in the unfiltered dataset, and masked probes were removed. This removal was followed by further removal of probes that did not pass previous QC, and that had not been removed by pOOBAH. Therefore, SeSAMe 2 has two rounds of probe removal. Noob normalization with nonlinear dye bias correction was then carried out in the filtered dataset. Methods were then compared by subsetting the 16 replicated samples and evaluating the effects that the different normalization methods had in the absolute difference of beta values (|β|) between replicated samples.

Genome-wide analysis of gene expression or protein binding patterns using different array or sequencing based technologies is now routinely performed to compare different populations, such as treatment and reference groups. It is often necessary to normalize the data obtained to remove technical variation introduced in the course of conducting experimental work, but standard normalization techniques are not capable of eliminating technical bias in cases where the distribution of the truly altered variables is skewed, i.e. when a large fraction of the variables are either positively or negatively affected by the treatment. However, several experiments are likely to generate such skewed distributions, including ChIP-chip experiments for the study of chromatin, gene expression experiments for the study of apoptosis, and SNP-studies of copy number variation in normal and tumour tissues. A preliminary study using spike-in array data established that the capacity of an experiment to identify altered variables and generate unbiased estimates of the fold change decreases as the fraction of altered variables and the skewness increases. We propose the following work-flow for analyzing high-dimensional experiments with regions of altered variables: (1) Pre-process raw data using one of the standard normalization techniques. (2) Investigate if the distribution of the altered variables is skewed. (3) If the distribution is not believed to be skewed, no additional normalization is needed. Otherwise, re-normalize the data using a novel HMM-assisted normalization procedure. (4) Perform downstream analysis. Here, ChIP-chip data and simulated data were used to evaluate the performance of the work-flow. It was found that skewed distributions can be detected by using the novel DSE-test (Detection of Skewed Experiments). Furthermore, applying the HMM-assisted normalization to experiments where the distribution of the truly altered variables is skewed results in considerably higher sensitivity and lower bias than can be attained using standard and invariant normalization methods.

A simple and robust non-linear method is presented for normalization using array signal distribution analysis and cubic splines. Both the regression and spline-based methods described performed better than existing linear methods when assessed on the variability of replicate arrays

Normalization is an essential step with considerable impact on high-throughput RNA sequencing (RNA-seq) data analysis. Although there are numerous methods for read count normalization, it remains a challenge to choose an optimal method due to multiple factors contributing to read count variability that affects the overall sensitivity and specificity. In order to properly determine the most appropriate normalization methods, it is critical to compare the performance and shortcomings of a representative set of normalization routines based on different dataset characteristics. Therefore, we set out to evaluate the performance of the commonly used methods (DESeq, TMM-edgeR, FPKM-CuffDiff, TC, Med UQ and FQ) and two new methods we propose: Med-pgQ2 and UQ-pgQ2 (per-gene normalization after per-sample median or upper-quartile global scaling). Our per-gene normalization approach allows for comparisons between conditions based on similar count levels. Using the benchmark Microarray Quality Control Project (MAQC) and simulated datasets, we performed differential gene expression analysis to evaluate these methods. When evaluating MAQC2 with two replicates, we observed that Med-pgQ2 and UQ-pgQ2 achieved a slightly higher area under the Receiver Operating Characteristic Curve (AUC), a specificity rate > 85%, the detection power > 92% and an actual false discovery rate (FDR) under 0.06 given the nominal FDR (≤0.05). Although the top commonly used methods (DESeq and TMM-edgeR) yield a higher power (>93%) for MAQC2 data, they trade off with a reduced specificity (

Background Affymetrix oligonucleotide arrays simultaneously measure the abundances of thousands of mRNAs in biological samples. Comparability of array results is necessary for the creation of large-scale gene expression databases. The standard strategy for normalizing oligonucleotide array readouts has practical drawbacks. We describe alternative normalization procedures for oligonucleotide arrays based on a common pool of known biotin-labeled cRNAs spiked into each hybridization. Results We first explore the conditions for validity of the 'constant mean assumption', the key assumption underlying current normalization methods. We introduce 'frequency normalization', a 'spike-in'-based normalization method which estimates array sensitivity, reduces background noise and allows comparison between array designs. This approach does not rely on the constant mean assumption and so can be effective in conditions where standard procedures fail. We also define 'scaled frequency', a hybrid normalization method relying on both spiked transcripts and the constant mean assumption while maintaining all other advantages of frequency normalization. We compare these two procedures to a standard global normalization method using experimental data. We also use simulated data to estimate accuracy and investigate the effects of noise. We find that scaled frequency is as reproducible and accurate as global normalization while offering several practical advantages. Conclusions Scaled frequency quantitation is a convenient, reproducible technique that performs as well as global normalization on serial experiments with the same array design, while offering several additional features. Specifically, the scaled-frequency method enables the comparison of expression measurements across different array designs, yields estimates of absolute message abundance in cRNA and determines the sensitivity of individual arrays.

Metagenomic time-course studies provide valuable insights into the dynamics of microbial systems and have become increasingly popular alongside the reduction in costs of next-generation sequencing technologies. Normalization is a common but critical preprocessing step before proceeding with downstream analysis. To the best of our knowledge, currently there is no reported method to appropriately normalize microbial time-series data. We propose TimeNorm, a novel normalization method that considers the compositional property and time dependency in time-course microbiome data. It is the first method designed for normalizing time-series data within the same time point (intra-time normalization) and across time points (bridge normalization), separately. Intra-time normalization normalizes microbial samples under the same condition based on common dominant features. Bridge normalization detects and utilizes a group of most stable features across two adjacent time points for normalization. Through comprehensive simulation studies and application to a real study, we demonstrate that TimeNorm outperforms existing normalization methods and boosts the power of downstream differential abundance analysis.

Introduction: MicroRNAs are small noncoding RNAs with potential regulatory roles in hypertension and drug response. The presence of many of these RNAs in biofluids has spurred investigation into their role as possible biomarkers for use in precision approaches to healthcare. One of the major challenges in clinical translation of circulating miRNA biomarkers is the limited replication across studies due to lack of standards for data normalization techniques for array-based approaches and a lack of consensus on an endogenous control normalizer for qPCR-based candidate miRNA profiling studies.Methods: We conducted genome-wide profiling of 754 miRNAs in baseline plasma of 36 European American individuals with uncomplicated hypertension selected from the PEAR clinical trial, who had been untreated for hypertension for at least one month prior to sample collection. After appropriate quality control with amplification score and missingness filters, we tested different normalization strategies such as normalization with global mean of imputed and unimputed data, mean of restricted set of miRNAs, quantile normalization, and endogenous control miRNA normalization to identify the method that best reduces the technical/experimental variability in the data. We identified best endogenous control candidates with expression pattern closest to the mean miRNA expression in the sample, as well as by assessing their stability using a combination of NormFinder, geNorm, Best Keeper and Delta Ct algorithms under the Reffinder software. The suitability of the four best endogenous controls was validated in 50 hypertensive African Americans from the same trial with reverse-transcription–qPCR and by evaluating their stability ranking in that cohort.Results: Among the compared normalization strategies, quantile normalization and global mean normalization performed better than others in terms of reducing the standard deviation of miRNAs across samples in the array-based data. Among the four strongest candidate miRNAs from our selection process (miR-223-3p, 19b, 106a, and 126-5p), miR-223-3p and miR-126-5p were consistently expressed with the best stability ranking in the validation cohort. Furthermore, the combination of miR-223-3p and 126-5p showed better stability ranking when compared to single miRNAs.Conclusion: We identified quantile normalization followed by global mean normalization to be the best methods in reducing the variance in the data. We identified the combination of miR-223-3p and 126-5p as potential endogenous control in studies of hypertension.

A robust semi-parametric normalization technique has been developed, based on the assumption that the large majority of genes will not have their relative expression levels changed from one treatment group to the next, and on the assumption that departures of the response from linearity are small and slowly varying. The method was tested using data simulated under various error models and it performs well.

Data normalization is a crucial step in the gene expression analysis as it ensures the validity of its downstream analyses. Although many metrics have been designed to evaluate the existing normalization methods, different metrics or different datasets by the same metric yield inconsistent results, particularly for the single-cell RNA sequencing (scRNA-seq) data. The worst situations could be that one method evaluated as the best by one metric is evaluated as the poorest by another metric, or one method evaluated as the best using one dataset is evaluated as the poorest using another dataset. Here raises an open question: principles need to be established to guide the evaluation of normalization methods. In this study, we propose a principle that one normalization method evaluated as the best by one metric should also be evaluated as the best by another metric (the consistency of metrics) and one method evaluated as the best using scRNA-seq data should also be evaluated as the best using bulk RNA-seq data or microarray data (the consistency of datasets). Then, we designed a new metric named Area Under normalized CV threshold Curve (AUCVC) and applied it with another metric mSCC to evaluate 14 commonly used normalization methods using both scRNA-seq data and bulk RNA-seq data, satisfying the consistency of metrics and the consistency of datasets. Our findings paved the way to guide future studies in the normalization of gene expression data with its evaluation. The raw gene expression data, normalization methods, and evaluation metrics used in this study have been included in an R package named NormExpression. NormExpression provides a framework and a fast and simple way for researchers to select the best method for the normalization of their gene expression data based on the evaluation of different methods (particularly some data-driven methods or their own methods) in the principle of the consistency of metrics and the consistency of datasets.

A number of procedures for normalization and detection of differentially expressed genes have been proposed. Four different normalization methods and all possible combinations with three different statistical algorithms have been used for detection of differentially expressed genes on a dataset. The number of genes detected as differentially expressed differs by a factor of about three.

The technological advances in mass spectrometry allow us to collect more comprehensive data with higher quality and increasing speed. With the rapidly increasing amount of data generated, the need for streamlining analyses becomes more apparent. Proteomics data is known to be often affected by systemic bias from unknown sources, and failing to adequately normalize the data can lead to erroneous conclusions. To allow researchers to easily evaluate and compare different normalization methods via a user-friendly interface, we have developed “proteiNorm”. The current implementation of proteiNorm accommodates preliminary filters on peptide and sample levels followed by an evaluation of several popular normalization methods and visualization of the missing value. The user then selects an adequate normalization method and one of the several imputation methods used for the subsequent comparison of different differential expression methods and estimation of statistical power. The application of proteiNorm and interpretation of its results are demonstrated on two tandem mass tag multiplex (TMT6plex and TMT10plex) and one label-free spike-in mass spectrometry example data set. The three data sets reveal how the normalization methods perform differently on different experimental designs and the need for evaluation of normalization methods for each mass spectrometry experiment. With proteiNorm, we provide a user-friendly tool to identify an adequate normalization method and to select an appropriate method for differential expression analysis.

Dataset Title: Data and Code for: "Universal Adaptive Normalization Scale (AMIS): Integration of Heterogeneous Metrics into a Unified System" Description: This dataset contains source data and processing results for validating the Adaptive Multi-Interval Scale (AMIS) normalization method. Includes educational performance data (student grades), economic statistics (World Bank GDP), and Python implementation of the AMIS algorithm with graphical interface. Contents: - Source data: educational grades and GDP statistics - AMIS normalization results (3, 5, 9, 17-point models) - Comparative analysis with linear normalization - Ready-to-use Python code for data processing Applications: - Educational data normalization and analysis - Economic indicators comparison - Development of unified metric systems - Methodology research in data scaling Technical info: Python code with pandas, numpy, scipy, matplotlib dependencies. Data in Excel format.

Based on open access data, 79 Mediterranean passenger ports are analyzed to compare their infrastructure, hinterland accessibility and offered multi-modality. Comparative Geo-spatial analysis is also carried out by using the data normalization method in order to visualize the ports' performance on maps. These data driven comprehensive analytical results can bring added value to sustainable development policy and planning initiatives in the Mediterranean Region. The analyzed elements can be also contributed to the development of passenger port performance indicators. The empirical research methods used for the Mediterranean passenger ports can be replicated for transport nodes of any region around the world to determine their relative performance on selected criteria for improvement and planning.

The Mediterranean passenger ports were initially categorizing into cruise and ferry ports. The cruise ports were identified from the member list of the Association for the Mediterranean Cruise Ports (MedCruise), representing more than 80% of the cruise tourism activities per country. The identified cruise ports were mapped by selecting the corresponding geo-referenced ports from the map layer developed by the European Marine Observation and Data Network (EMODnet). The United Nations (UN) Code for Trade and Transport Locations (LOCODE) was identified for each of the cruise ports as the common criteria to carry out the selection. The identified cruise ports not listed by the EMODnet were added to the geo-database by using under license the editing function of the ArcMap (version 10.1) geographic information system software. The ferry ports were identified from the open access industry initiative data provided by the Ferrylines, and were mapped in a similar way as the cruise ports (Figure 1).

Based on the available data from the identified cruise ports, a database (see Table A1–A3) was created for a Mediterranean scale analysis. The ferry ports were excluded due to the unavailability of relevant information on selected criteria (Table 2). However, the cruise ports serving as ferry passenger ports were identified in order to maximize the scope of the analysis. Port infrastructure and hinterland accessibility data were collected from the recent statistical reports published by the MedCruise, which are a compilation of data provided by its individual member port authorities and the cruise terminal operators. Other supplementary sources were the European Sea Ports Organization (ESPO) and the Global Ports Holding, a cruise terminal operator with an established presence in the Mediterranean. Additionally, open access data sources (e.g. the Google Maps and Trip Advisor) were consulted in order to identify the multi-modal transports and bridge the data gaps on hinterland accessibility by measuring the approximate distances.

Label-free proteomics expression data sets often exhibit data heterogeneity and missing values, necessitating the development of effective normalization and imputation methods. The selection of appropriate normalization and imputation methods is inherently data-specific, and choosing the optimal approach from the available options is critical for ensuring robust downstream analysis. This study aimed to identify the most suitable combination of these methods for quality control and accurate identification of differentially expressed proteins. In this study, we developed nine combinations by integrating three normalization methods, locally weighted linear regression (LOESS), variance stabilization normalization (VSN), and robust linear regression (RLR) with three imputation methods: k-nearest neighbors (k-NN), local least-squares (LLS), and singular value decomposition (SVD). We utilized statistical measures, including the pooled coefficient of variation (PCV), pooled estimate of variance (PEV), and pooled median absolute deviation (PMAD), to assess intragroup and intergroup variation. The combinations yielding the lowest values corresponding to each statistical measure were chosen as the data set’s suitable normalization and imputation methods. The performance of this approach was tested using two spiked-in standard label-free proteomics benchmark data sets. The identified combinations returned a low NRMSE and showed better performance in identifying spiked-in proteins. The developed approach can be accessed through the R package named ’lfproQC’ and a user-friendly Shiny web application (https://dabiniasri.shinyapps.io/lfproQC and http://omics.icar.gov.in/lfproQC), making it a valuable resource for researchers looking to apply this method to their data sets.

This dataset is a project file generated by BMDExpress 2.2 SW (Sciome, Research Triangle Park, NC). It contains gene expression data for livers of rats exposed to 4 chemicals (crude MCHM, neat MCHM, DMPT, p-toluidine) and kidneys of rats exposed to PPH. The project file includes normalized expression data (GeneChip Rat 230 2.0 Array) using 7 different pre-processing methods (RMA, GCRMA, MAS5.0, MAS5.0_noA calls, PLIER, PLIER16, and PLIER16_noA calls); differentially expressed probe-sets detected by William's method (p<0.05, and minimum fold change of 1.5); probeset-level and pathway-level BMD and BMDL values from transcriptomic dose-response modeling. This dataset is associated with the following publication: Mezencev, R., and S. Auerbach. The sensitivity of transcriptomics BMD modeling to the methods used for microarray data normalization. PLOS ONE. Public Library of Science, San Francisco, CA, USA, 15(5): e0232955, (2020).

Simulation script 1This R script will simulate two populations of microbiome samples and compare normalization methods.Simulation script 2This R script will simulate two populations of microbiome samples and compare normalization methods via PcOAs.Sample.OTU.distributionOTU distribution used in the paper: Methods for normalizing microbiome data: an ecological perspective

Detailed spearman correlation coefficient results for all normalization methods. (XLSX 17Â kb)

This research study aims to understand the application of Artificial Neural Networks (ANNs) to forecast the Self-Compacting Recycled Coarse Aggregate Concrete (SCRCAC) compressive strength. From different literature, 602 available data sets from SCRCAC mix designs are collected, and the data are rearranged, reconstructed, trained and tested for the ANN model development. The models were established using seven input variables: the mass of cementitious content, water, natural coarse aggregate content, natural fine aggregate content, recycled coarse aggregate content, chemical admixture and mineral admixture used in the SCRCAC mix designs. Two normalization techniques are used for data normalization to visualize the data distribution. For each normalization technique, three transfer functions are used for modelling. In total, six different types of models were run in MATLAB and used to estimate the 28th day SCRCAC compressive strength. Normalization technique 2 performs better than 1 and TANSING is the best transfer function. The best k-fold cross-validation fold is k = 7. The coefficient of determination for predicted and actual compressive strength is 0.78 for training and 0.86 for testing. The impact of the number of neurons and layers on the model was performed. Inputs from standards are used to forecast the 28th day compressive strength. Apart from ANN, Machine Learning (ML) techniques like random forest, extra trees, extreme boosting and light gradient boosting techniques are adopted to predict the 28th day compressive strength of SCRCAC. Compared to ML, ANN prediction shows better results in terms of sensitive analysis. The study also extended to determine 28th day compressive strength from experimental work and compared it with 28th day compressive strength from ANN best model. Standard and ANN mix designs have similar fresh and hardened properties. The average compressive strength from ANN model and experimental results are 39.067 and 38.36 MPa, respectively with correlation coefficient is 1. It appears that ANN can validly predict the compressive strength of concrete.

The label-free quantitative mass spectrometry methods, in particular the SWATH-MS approach, have gained popularity and became a powerful technique for comparison of large datasets. In the present work, it is introduced the use of recombinant proteins as internal standards for untargeted label-free methods. The proposed internal standard strategy reveals a similar intragroup normalization capacity when compared with the most common normalization methods, with the additional advantage of maintaining the overall proteome changes between groups (which are lost using the methods referred above). Thus, proving to be able to maintain a good performance even when large qualitative and quantitative differences in sample composition are observed, such as the ones induced by biological regulation (as observed in secretome and other biofluids’ analyses) or by enrichment approaches (such as immunopurifications). Moreover, it corresponds to a cost-effective alternative, easier to implement than the current stable-isotope labeling internal standards, therefore being an appealing strategy for large quantitative screening, as clinical cohorts for biomarker discovery.

This dataset tracks the updates made on the dataset "A new non-linear normalization method for reducing variability in DNA microarray experiments" as a repository for previous versions of the data and metadata.