An Iterative Leave-One-Out Approach to Outlier Detection in RNA-Seq Data.
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ABSTRACT:
SUBMITTER: George NI
PROVIDER: S-EPMC4454687 | biostudies-literature | 2015
REPOSITORIES: biostudies-literature
Publications
An Iterative Leave-One-Out Approach to Outlier Detection in RNA-Seq Data.
George Nysia I NI Bowyer John F JF Crabtree Nathaniel M NM Chang Ching-Wei CW
PloS one 20150603 6
The discrete data structure and large sequencing depth of RNA sequencing (RNA-seq) experiments can often generate outlier read counts in one or more RNA samples within a homogeneous group. Thus, how to identify and manage outlier observations in RNA-seq data is an emerging topic of interest. One of the main objectives in these research efforts is to develop statistical methodology that effectively balances the impact of outlier observations and achieves maximal power for statistical testing. To ...[more]
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Project description:BACKGROUND:High throughput RNA sequencing is a powerful approach to study gene expression. Due to the complex multiple-steps protocols in data acquisition, extreme deviation of a sample from samples of the same treatment group may occur due to technical variation or true biological differences. The high-dimensionality of the data with few biological replicates make it challenging to accurately detect those samples, and this issue is not well studied in the literature currently. Robust statistics is a family of theories and techniques aim to detect the outliers by first fitting the majority of the data and then flagging data points that deviate from it. Robust statistics have been widely used in multivariate data analysis for outlier detection in chemometrics and engineering. Here we apply robust statistics on RNA-seq data analysis. RESULTS:We report the use of two robust principal component analysis (rPCA) methods, PcaHubert and PcaGrid, to detect outlier samples in multiple simulated and real biological RNA-seq data sets with positive control outlier samples. PcaGrid achieved 100% sensitivity and 100% specificity in all the tests using positive control outliers with varying degrees of divergence. We applied rPCA methods and classical principal component analysis (cPCA) on an RNA-Seq data set profiling gene expression of the external granule layer in the cerebellum of control and conditional SnoN knockout mice. Both rPCA methods detected the same two outlier samples but cPCA failed to detect any. We performed differentially expressed gene detection before and after outlier removal as well as with and without batch effect modeling. We validated gene expression changes using quantitative reverse transcription PCR and used the result as reference to compare the performance of eight different data analysis strategies. Removing outliers without batch effect modeling performed the best in term of detecting biologically relevant differentially expressed genes. CONCLUSIONS:rPCA implemented in the PcaGrid function is an accurate and objective method to detect outlier samples. It is well suited for high-dimensional data with small sample sizes like RNA-seq data. Outlier removal can significantly improve the performance of differential gene detection and downstream functional analysis.
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Project description:Background. A common research goal in transcriptome projects is to find genes that are differentially expressed in different phenotype classes. Biologists might wish to validate such gene candidates experimentally, or use them for downstream systems biology analysis. Producing a coherent differential gene expression analysis from RNA-seq count data requires an understanding of how numerous sources of variation such as the replicate size, the hypothesized biological effect size, and the specific method for making differential expression calls interact. We believe an explicit demonstration of such interactions in real RNA-seq data sets is of practical interest to biologists. Results. Using two large public RNA-seq data sets-one representing strong, and another mild, biological effect size-we simulated different replicate size scenarios, and tested the performance of several commonly-used methods for calling differentially expressed genes in each of them. We found that, when biological effect size was mild, RNA-seq experiments should focus on experimental validation of differentially expressed gene candidates. Importantly, at least triplicates must be used, and the differentially expressed genes should be called using methods with high positive predictive value (PPV), such as NOISeq or GFOLD. In contrast, when biological effect size was strong, differentially expressed genes mined from unreplicated experiments using NOISeq, ASC and GFOLD had between 30 to 50% mean PPV, an increase of more than 30-fold compared to the cases of mild biological effect size. Among methods with good PPV performance, having triplicates or more substantially improved mean PPV to over 90% for GFOLD, 60% for DESeq2, 50% for NOISeq, and 30% for edgeR. At a replicate size of six, we found DESeq2 and edgeR to be reasonable methods for calling differentially expressed genes at systems level analysis, as their PPV and sensitivity trade-off were superior to the other methods'. Conclusion. When biological effect size is weak, systems level investigation is not possible using RNAseq data, and no meaningful result can be obtained in unreplicated experiments. Nonetheless, NOISeq or GFOLD may yield limited numbers of gene candidates with good validation potential, when triplicates or more are available. When biological effect size is strong, NOISeq and GFOLD are effective tools for detecting differentially expressed genes in unreplicated RNA-seq experiments for qPCR validation. When triplicates or more are available, GFOLD is a sharp tool for identifying high confidence differentially expressed genes for targeted qPCR validation; for downstream systems level analysis, combined results from DESeq2 and edgeR are useful.
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Project description:To design an algorithm for detecting outliers over streaming data has become an important task in many common applications, arising in areas such as fraud detections, network analysis, environment monitoring and so forth. Due to the fact that real-time data may arrive in the form of streams rather than batches, properties such as concept drift, temporal context, transiency, and uncertainty need to be considered. In addition, data processing needs to be incremental with limited memory resource, and scalable. These facts create big challenges for existing outlier detection algorithms in terms of their accuracies when they are implemented in an incremental fashion, especially in the streaming environment. To address these problems, we first propose C_KDE_WR, which uses sliding window and kernel function to process the streaming data online, and reports its results demonstrating high throughput on handling real-time streaming data, implemented in a CUDA framework on Graphics Processing Unit (GPU). We also present another algorithm, C_LOF, based on a very popular and effective outlier detection algorithm called Local Outlier Factor (LOF) which unfortunately works only on batched data. Using a novel incremental approach that compensates the drawback of high complexity in LOF, we show how to implement it in a streaming context and to obtain results in a timely manner. Like C_KDE_WR, C_LOF also employs sliding-window and statistical-summary to help making decision based on the data in the current window. It also addresses all those challenges of streaming data as addressed in C_KDE_WR. In addition, we report the comparative evaluation on the accuracy of C_KDE_WR with the state-of-the-art SOD_GPU using Precision, Recall and F-score metrics. Furthermore, a t-test is also performed to demonstrate the significance of the improvement. We further report the testing results of C_LOF on different parameter settings and drew ROC and PR curve with their area under the curve (AUC) and Average Precision (AP) values calculated respectively. Experimental results show that C_LOF can overcome the masquerading problem, which often exists in outlier detection on streaming data. We provide complexity analysis and report experiment results on the accuracy of both C_KDE_WR and C_LOF algorithms in order to evaluate their effectiveness as well as their efficiencies.
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Project description:BackgroundAs a powerful tool, RNA-Seq has been widely used in various studies. Usually, unmapped RNA-seq reads have been considered as useless and been trashed or ignored.ResultsWe develop a strategy to mining the full length sequence by unmapped reads combining with specific reverse transcription primers design and high throughput sequencing. In this study, we salvage 36 unmapped reads from standard RNA-Seq data and randomly select one 149 bp read as a model. Specific reverse transcription primers are designed to amplify its both ends, followed by next generation sequencing. Then we design a statistical model based on power law distribution to estimate its integrality and significance. Further, we validate it by Sanger sequencing. The result shows that the full length is 1556 bp, with insertion mutations in microsatellite structure.ConclusionWe believe this method would be a useful strategy to extract the sequences information from the unmapped RNA-seq data. Further, it is an alternative way to get the full length sequence of unknown cDNA.
| S-EPMC8626966 | biostudies-literature