Missing values are common in high-throughput mass spectrometry data. Two strategies are available to address missing values: (i) eliminate or impute the missing values and apply statistical methods that require complete data and (ii) use statistical methods that specifically account for missing values without imputation (imputation-free methods). This study reviews the effect of sample size and percentage of missing values on statistical inference for multiple methods under these two strategies. With increasing missingness, the ability of imputation and imputation-free methods to identify differentially and non-differentially regulated compounds in a two-group comparison study declined. Random forest and k-nearest neighbor imputation combined with a Wilcoxon test performed well in statistical testing for up to 50% missingness with little bias in estimating the effect size. Quantile regression imputation accompanied with a Wilcoxon test also had good statistical testing outcomes but substantially distorted the difference in means between groups. None of the imputation-free methods performed consistently better for statistical testing than imputation methods.

With expanded access to, and decreased costs of, mass spectrometry, investigators are collecting and analyzing multiple biological matrices from the same subject such as serum, plasma, tissue and urine to enhance biomarker discoveries, understanding of disease processes and identification of therapeutic targets. Commonly, each biological matrix is analyzed separately, but multivariate methods such as MANOVAs that combine information from multiple biological matrices are potentially more powerful. However, mass spectrometric data typically contain large amounts of missing values, and imputation is often used to create complete data sets for analysis. The effects of imputation on multiple biological matrix analyses have not been studied. We investigated the effects of seven imputation methods (half minimum substitution, mean substitution, k-nearest neighbors, local least squares regression, Bayesian principal components analysis, singular value decomposition and random forest), on the within-subject correlation of compounds between biological matrices and its consequences on MANOVA results. Through analysis of three real omics data sets and simulation studies, we found the amount of missing data and imputation method to substantially change the between-matrix correlation structure. The magnitude of the correlations was generally reduced in imputed data sets, and this effect increased with the amount of missing data. Significant results from MANOVA testing also were substantially affected. In particular, the number of false positives increased with the level of missing data for all imputation methods. No one imputation method was universally the best, but the simple substitution methods (Half Minimum and Mean) consistently performed poorly.

MOTIVATION: High through-put mass spectrometry (MS) is now being used to profile small molecular compounds across multiple biological sample types from the same subjects with the goal of leveraging information across biospecimens. Multivariate statistical methods that combine information from all biospecimens could be more powerful than the usual univariate analyses. However, missing values are common in MS data and imputation can impact between-biospecimen correlation and multivariate analysis results. RESULTS: We propose two multivariate two-part statistics that accommodate missing values and combine data from all biospecimens to identify differentially regulated compounds. Statistical significance is determined using a multivariate permutation null distribution. Relative to univariate tests, the multivariate procedures detected more significant compounds in three biological datasets. In a simulation study, we showed that multi-biospecimen testing procedures were more powerful than single-biospecimen methods when compounds are differentially regulated in multiple biospecimens but univariate methods can be more powerful if compounds are differentially regulated in only one biospecimen. AVAILABILITY AND IMPLEMENTATION: We provide R functions to implement and illustrate our method as supplementary information CONTACT: [email protected] information: Supplementary data are available at Bioinformatics online.

BACKGROUND: High-density microarray technology is increasingly applied to study gene expression levels on a large scale. Microarray experiments rely on several critical steps that may introduce error and uncertainty in analyses. These steps include mRNA sample extraction, amplification and labeling, hybridization, and scanning. In some cases this may be manifested as systematic spatial variation on the surface of microarray in which expression measurements within an individual array may vary as a function of geographic position on the array surface. RESULTS: We hypothesized that an index of the degree of spatiality of gene expression measurements associated with their physical geographic locations on an array could indicate the summary of the physical reliability of the microarray. We introduced a novel way to formulate this index using a statistical analysis tool. Our approach regressed gene expression intensity measurements on a polynomial response surface of the microarray's Cartesian coordinates. We demonstrated this method using a fixed model and presented results from real and simulated datasets. CONCLUSION: We demonstrated the potential of such a quantitative metric for assessing the reliability of individual arrays. Moreover, we showed that this procedure can be incorporated into laboratory practice as a means to set quality control specifications and as a tool to determine whether an array has sufficient quality to be retained in terms of spatial correlation of gene expression measurements.

BACKGROUND: Many efforts in microarray data analysis are focused on providing tools and methods for the qualitative analysis of microarray data. HDBStat! (High-Dimensional Biology-Statistics) is a software package designed for analysis of high dimensional biology data such as microarray data. It was initially developed for the analysis of microarray gene expression data, but it can also be used for some applications in proteomics and other aspects of genomics. HDBStat! provides statisticians and biologists a flexible and easy-to-use interface to analyze complex microarray data using a variety of methods for data preprocessing, quality control analysis and hypothesis testing. RESULTS: Results generated from data preprocessing methods, quality control analysis and hypothesis testing methods are output in the form of Excel CSV tables, graphs and an Html report summarizing data analysis. CONCLUSION: HDBStat! is a platform-independent software that is freely available to academic institutions and non-profit organizations. It can be downloaded from our website http://www.soph.uab.edu/ssg_content.asp?id=1164.

BACKGROUND: A typical microarray experiment has many sources of variation which can be attributed to biological and technical causes. Identifying sources of variation and assessing their magnitude, among other factors, are important for optimal experimental design. The objectives of this study were: (1) to estimate relative magnitudes of different sources of variation and (2) to evaluate agreement between biological and technical replicates. RESULTS: We performed a microarray experiment using a total of 24 Affymetrix GeneChip arrays. The study included 4th mammary gland samples from eight 21-day-old Sprague Dawley CD female rats exposed to genistein (soy isoflavone). RNA samples from each rat were split to assess variation arising at labeling and hybridization steps. A general linear model was used to estimate variance components. Pearson correlations were computed to evaluate agreement between technical and biological replicates. CONCLUSION: The greatest source of variation was biological variation, followed by residual error, and finally variation due to labeling when *.cel files were processed with dChip and RMA image processing algorithms. When MAS 5.0 or GCRMA-EB were used, the greatest source of variation was residual error, followed by biology and labeling. Correlations between technical replicates were consistently higher than between biological replicates.