Revealing MicroRNA Biomarkers for Alzheimer’s Disease Using Next Generation Sequencing Data



Table of content

  1. Abstract
  2. Related works
  3. Methodology
  4. Results and Analysis
  5. Conclusion
  6. Publications
  7. Links


Alzheimer’s disease is recognized as one of the common diseases found among older people, which still has no successful cure. In this study, our goal is to determine the best set of miRNA biomarkers which are highly differentially expressed in Alzheimer’s disease. Using statistical analysis followed by machine learning techniques, we establish 25 microRNAs as biomarkers for AD. Furthermore, we provide an analysis of the selected 25 microRNAs with area under the receiver operating curve and classification algorithms.

Sample selection

When detecting biomarkers for Alzheimer’s disease, initially we have to select a sample for performing analysis. Mostly blood samples are used due to the high availability. Different types of blood samples including whole blood, serum and plasma are used by many previous researchers where they have tried to find miRNA biomarkers. If we use brain samples it would give most accurate results than blood samples since AD is most prominently active in brain. We would be able to give more accurate results if both blood and brain samples are used. Samples can be taken from participants generally as, AD and controls and also they can be taken considering the different stages as severe, moderate, mild AD and controls. Another approach in collecting samples is taking then from participants with HC, MCI and AD. The number of samples used when developing a diagnosis method can be identified as one of the main factors which could affect the final results. Next generation sequencing platform is the most trending method used for gathering samples for various disease diagnosis researches. Many techniques like Illumina sequencing technique are introduced for working with NGS data. Preprocessing the raw sequence counts can be done using a bioinformatic pipeline, which gives the read counts for each miRNA as the final outcome.


Normalization of sequencing read counts can be performed using several normalization methods. Quantile normalization is one way that we can do normalization when we are having a high dimensional dataset. It excludes selected samples to minimize noise. Mean normalized read counts also can be used to filter out the miRNAs. Also we can follow a stepwise procedure to do normalization as below.

Statistical Analysis

Initial detection of miRNA can be done by initially calculating a significance value(p value). P value is a value between 0 and 1, which shows the level of statistical significance. If a p value is less than the significance level (0.05), it is considered as a nominally significant p value and we can select those miRNA as the most impacting miRNAs.

WMW test, Wald test and Fisher’s exact test can be used to calculate the p values and these p values can be adjusted for multiple testing using an approach like Benjamini-Hochberg approach. Other than that, t test and kruskal test can also be used to calculate significance values.

Validation of samples

Validation of the samples makes it easier for the next steps in the investigation and also it makes the final results more accurate. After the statistical analysis process, for validating the obtained samples, quantitative real time-polymerase chain reaction (qRT-PCR) method is used by many researchers. It analyzes the expression of single miRNAs by applying the method on previously used samples for sequencing. But in a previous study, they have additionally included patients with AD and also patients with other neurological disorders in the validation step, to analyze the the set of miRNAs they obtained in the previous step. After the validation is carried out, the miRNAs can be further filtered out to obtain the most significant miRNAs.

Receiver operating characteristic curves

Receiver operating characteristic curve analysis is used to evaluate the performance or accuracy of a classification model. ROC is a plot of sensitivity against specificity for selected samples. It is also used to initially detect the dysregulation of miRNAs and to discriminate between AD and NC sample groups. The area under the curve is the degree of separability. If the AUC is high, that means that particular miRNA is better to distinguish patients with AD and control.

Feature selection and Classification

If we use a classification model without using feature selection, it will take more run time due to the huge size with redundant features. Therefore it is required to apply some feature selection method to reduce those redundant features. Hierarchical clustering is a feature selection method which can be used to statistically analyze the dataset. It will build clusters of miRNAs having similar patterns. Principal Component Analysis is another approach which can be used for the feature selection. Machine learning classifier models are used to predict whether a sample belongs to AD or control. AdaboostM1, J48 decision tree, random forest and support vector machines and radial basis SVM are some machine learning approaches that can be used for building prediction models. In a previously done study, they have built a separate model by performing 7-way cross validation using 7 randomly picked partitions of 5 positive and 5 negative samples each for the feature selection.


According to the review we have done, we identified how we can use miRNAs to diagnosis AD and what are the miRNA diagnostic biomarkers which can be found in AD patients. In each study, for filtering out the candidate miRNA, step wise procedures including initial detection and statistical analysis have performed. When consider about previous studies, there are several limitations. The most common limitation of most of the research is they used a limited number of the cohort to their experiments. It is hard to find a large number of Alzheimer’s disease patients to do massive experiments. But we can obtain better results if we expand the cohort size. In many studies, samples with analyzed dementia and controls have used. But not discussing about the possibility to discover pre-clinical biomarkers for Alzheimer disease is a limitation of most of the previous studies. A model which was built in a one previously done study, does not develop to anticipate movement from HC to MCI or MCI to AD. Also, this model was incapable of applying for late-stage AD findings. In another study, they have mentioned that they were unable to recognize a mechanism to identify the variation of miRNAs in serum samples. Considering all the drawbacks, limitations and also the developments found in the previous studies, in this research, we are focusing on finding a more accurate solution for detecting AD biomarkers.

Following Figure shows a summary of different methods used andthe results obtained in previous studies. According to this diagram, only 4 studies haveused machine learning algorithms and only 5 studies have used statistical methods intheir studies. Out of the results obtained from above mentioned 9 studies, 7 miRNAswere identified as common for those 9 studies.

Figure 02


Data collection

We used a data set available in National Center for Biotechnology Information (NCBI) database under the access number GSE46579. It includes 70 samples with 22 control and 48 AD and 2652 miRNAs.


The Next Generation Sequencing data preprocessing was done using the Galaxy platform. Galaxy is a web-based, opensource platform for scientific data analysis. First, the quality report of sequencing data was generated using the FastQC tool. Then, using the tool Trim Galore, data trimming was performed. The package Trim Galore allows both quality trimming and adapter trimming at once. Low-quality reads and adapters were removed from sequence read in the trimming procedure. Trimming increases the quality of sequences. Next, the data filtering procedure was performed using the Filter FASTQ tool. Short read sequences and low-quality sequences were removed in filtering. After that, the NGS reads were mapped against a reference genome (h38) using Bowtie2. Bowtie2 tool aligns sequences to the long reference sequences. Then, the reads were mapped against the hsa.gff3 miRNA precursor sequences from the miRBase database (v22) and the number of read counts of each miRNA was found using the htseq-count tool. This preprocessing procedure was done for every sample using the galaxy platform and then the summarized dataset was created with miRNA read counts of each sample. For the analysis purposes we used a data set with highly abundant miRNAs. To do that we considered miRNAs with read counts less than 50 across all samples of AD and control separately, as lowly abundant and removed them from the data set. Considering the mean distribution of the data set, we normalized the data set using quantile normalization technique instead of general normalization technique.

Statistical Analysis

Normalized data set obtained from the previous stage was further analyzed with significance value and fold change to reduce the number of features. We calculated the pValues for each miRNA using Wilicoxon-Mann-Whitney (WMW) test. Generally, fold change is a technique which is used to get an idea of how much change occurs going from one value to another. In this project we tried to get fold change values (log2) for each miRNA, to check the significant changes of each miRNA across AD and control samples. We used cut off values for pValues and fold changes values as 0.05 and 1 respectively, to obtain the highly expressed set of miRNAs. For each of those filtered features, we calculated the AUC values. Using those AUC values, another set of features were filtered out. Features with AUC score less than or equal to 0.5 were ignored as they don’t make a significant impact on the classification of the data set.

Feature Selection

Initially we used two different methods as PCA and Random Forest for selecting the best set of features. For the data set we obtained from the previous stage, we separately did PCA analysis and Random Forest analysis. Univariate feature selection method was used to decide how many features we needed to select from each. Features which have a significant relationship with the class value were identified from this univariate feature selection method. Next, the set of overlapped miRNAs from those two methods was identified as the best set of features which could be obtained from this part of feature selection. In the next part of the feature selection stage, we used correlation coefficient. As the correlation coefficient, we used Pearson correlation coefficient.

Figure 01


Classification accuracy was used to see how accurate our predictions were. A set of machine learning algorithms were modelled for the initial data set and out of those the most accurate algorithms were identified. Those each pre-identified algorithms were used for obtaining the classification accuracy of the final data set with biomarker miRNAs.


For validating the results we used Human MiRNA Disease Database version 3.2 (HMDD v3.2). HMDD contains a large set of miRNAs and related diseases collected from the literature. There are 35547 miRNA-disease associations in version 3.2 and it includes 1206 miRNAs, 893 diseases from 19280 related publications.

Results and Analysis

At the end of the preprocessing stage, a data set with 513 highly abundant miRNAs was obtained after removing miRNAs with less than 50 read counts across all samples. Considering the cut-off significance value as 0.05 and fold change (log2) as 1, the number of features were reduced up to 228. With the AUC analysis we further reduced the number of miRNAs and at the end of the statistical analysis, we identified 219 miRNAs as a set of highly expressed miRNAs. From the univariate feature selection method, we identified 50 miRNAs which have a significance relationship with class value. We identified 14 common miRNAs from two sets of miRNAs selected from PCA and random forest analysis. They are hsa-miR-186-5p, hsa-miR-144-3p, hsa-miR-151a-3p, hsa-miR-99b-5p, hsa-miR-98, hsa-miR-148a-3p, hsa-let-7g-5p, hsa-let-7f-5p, hsa-let-7a-5p, hsa-miR-30d-5p, hsa-miR-15a-5p, hsa-miR-589-5p, hsa-miR-144-5p, and hsa-let-7f-5p. We used heat maps for making a judgement on the correlation of each features obtained from previously mentioned two methods. Figure 2 and Figure 3 show how different features obtained from PCA and Random Forest analysis, are correlated. With the help of the heat maps, we decided to use 36 less correlated features for further analysis using correlation coefficients. Out of the different machine learning algorithms which we modelled for our initial data set, we identified the three most accurate algorithms namely, Support Vector Machine, Logistic Regression and Random Forest. For the data set with the 36 miRNAs, we calculated classification accuracy using those three models by varying the correlation coefficients. For each model we identified a correlation coefficient which gave the highest accuracy. Fig. 4 shows the plots with correlation coefficient against the classification accuracy of three models The three correlation coefficients were 0.9975, 0.5875 and 0.5300 for SVM, Logistic Regression and Random Forest respectively. Using those 3 correlation coefficients, three sub sets of miRNAs were obtained from the earlier used 36. As we calculated classification accuracy for the previously mentioned three subsets, we identified 11 miRNAs which provided the highest classification accuracy. They are, hsa-miR-4781-3p, brain-miR-112, hsa-let-7a-5p, hsa-miR-148b-5p, hsa-miR-29b-3p, brain-miR-431, hsa-miR-378a-5p, hsa-miR-548h-5p, hsa-miR-3909, hsa-miR-625-5p, and hsa-miR-24-3p.


In this report we have discussed about how to detect miRNA biomarkers for Alzheimer’s disease using next generation sequencing. Initially we have discussed about the need of a solution to identify Alzheimer’s disease in the early stage. Then we have mentioned about the literature review we have done. When we were doing the literature review, we have identified several miRNA biomarkers in different studies which used NGS. In these studies there were some limitations. In our approach so far, initially we have taken samples from participants with AD and control. Then samples were preprocessed and statistically analyzed. Significance values were calculated using Wilcoxon-Mann-Whitney (WMW) test. Also we have used ROC analysis. Using this procedure, here we have identified a set of significant miRNAs for AD. Using PCA, Random Forests and Correlation coefficient we identified 25 biomarker miRNAs for AD. In the next phase we validated the the result using HMDD v3.2. Leidinger et al., who have carried out a different method to find biomarkers using the same data set, have stated that they have obtained an accuracy of 93.3\% where we obtained an accuracy of 95.24\%. Addition to that we evaluated the results with specificity, sensitivity and AUC values as discussed previously. In addition to diagnosis of AD patients with the final set of biomarkers, the followed methodology can be used to identify different cures for other neurological diseases including AD, by effortlessly analyzing various data sets.


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