miRFam: an effective automatic miRNA classification method based on n-grams and a multiclass SVM
© Ding et al; licensee BioMed Central Ltd. 2011
Received: 29 September 2010
Accepted: 28 May 2011
Published: 28 May 2011
MicroRNAs (miRNAs) are ~22 nt long integral elements responsible for post-transcriptional control of gene expressions. After the identification of thousands of miRNAs, the challenge is now to explore their specific biological functions. To this end, it will be greatly helpful to construct a reasonable organization of these miRNAs according to their homologous relationships. Given an established miRNA family system (e.g. the miRBase family organization), this paper addresses the problem of automatically and accurately classifying newly found miRNAs to their corresponding families by supervised learning techniques. Concretely, we propose an effective method, miRFam, which uses only primary information of pre-miRNAs or mature miRNAs and a multiclass SVM, to automatically classify miRNA genes.
An existing miRNA family system prepared by miRBase was downloaded online. We first employed n-grams to extract features from known precursor sequences, and then trained a multiclass SVM classifier to classify new miRNAs (i.e. their families are unknown). Comparing with miRBase's sequence alignment and manual modification, our study shows that the application of machine learning techniques to miRNA family classification is a general and more effective approach. When the testing dataset contains more than 300 families (each of which holds no less than 5 members), the classification accuracy is around 98%. Even with the entire miRBase15 (1056 families and more than 650 of them hold less than 5 samples), the accuracy surprisingly reaches 90%.
Based on experimental results, we argue that miRFam is suitable for application as an automated method of family classification, and it is an important supplementary tool to the existing alignment-based small non-coding RNA (sncRNA) classification methods, since it only requires primary sequence information.
The source code of miRFam, written in C++, is freely and publicly available at: http://admis.fudan.edu.cn/projects/miRFam.htm.
Sequences of DNA, RNA and proteins are the fundamental currency of modern biological research, which link the different levels of the biological hierarchy, from genes to 3D structures . Common features of species and functionally important residues can be identified through sequence mining. RNA, which stores information like DNA and acts as an enzyme like proteins, may have supported cellular or pre-cellular life , and is crucial to protein synthesis that plays a very important role in life.
MiRBase is the central online repository of miRNA nomenclature, sequence data, annotation and target prediction, which first appeared in Oct. 2002 . Release 15 contains 14197 miRNA loci from 66 species. From version 5.0, miRBase began to classify miRNAs into different families.
This kind of information was stored in miFam.dat, which was freely available online http://www.mirbase.org. These families were prepared manually. Essentially, it was done by using the single-linkage method to cluster the precursor sequences based on BLAST hits, and then adjusting (merging and/or splitting) manually the clustered families by multiple sequence alignment. The aim is to put miRNAs that have a common ancestor into the same family.
Rfam  is another well known RNA database. It contains a collection of multiple sequence alignments and covariance models (CMs) that represent ncRNA families. The primary aim of Rfam is to annotate new members of known RNA families on nucleotide sequences, particularly complete genomes, by using sensitive BLAST filters in combination with CMs. Both primary sequences and base-paired secondary structures are used to establish and annotate families. Release 10 contains 1446 families, including 453 miRNA families. But the quality of multiple sequence alignments and secondary structures is still a challenge for Rfam. Furthermore, Rfam requires a lot of computing resources to establish the family structure, which is time consuming, especially when the number of sequences is huge.
Since pre-miRNAs can form stable hairpins, this specific structural property has been used to cluster or classify them by some ncRNA clustering or classification methods [23, 24]. Will et al. presented a structure-based clustering approach, LocARNA (local alignment of RNA), which is capable of extracting putative RNA classes from genome-wide survey of structured RNAs. The performance of LocARNA relies on the prediction accuracy of RNA secondary structures. However, current RNA secondary structure energy models are not always able to predict native RNA structures, even for short molecules . Furthermore, hairpin secondary structure might be less effective in miRNA classification since all miRNAs can fold back into this type of structure.
By far, multiple sequence and/or structure alignments are still widely used in ncRNA clustering and classification field. But neither of them has completely solved the ncRNA clustering or classification problem, especially for miRNAs. Not to mention effectiveness, only efficiency is still far from being satisfactory, since these methods could be very time-consuming when applied to large-scale validation of miRNA families.
As we know, miRNAs are highly conserved in not only their primary sequences but also their secondary structures. And miRNAs in the same family always have consensus secondary structures and similar functions . Hence, it is biologically significant to subsume miRNAs with consensus second structures and similar functions to the same family. In this paper, based on the family system provided by miRBase, we explored supervised learning techniques to accurately and automatically classify pre-miRNA or mature sequences.
Concretely, we propose an effective alignment free model named miRFam to classify newly detected miRNAs. First, it extracts n-grams as features from primary sequences. Then, these n-gram features are integrated into one feature vector by concentration. Finally, it trains a multiclass SVM classifier SVM multiclass based on the families prepared by miRBase to classify new pre-miRNA or mature sequences whose families are not yet known.
As a powerful tool, miRFam aims to classify new miRNAs into their corresponding families. It can not only support researchers who just obtained novel miRNAs computationally or experimentally to go on exploring the function of these miRNAs, but also enhance the utility of miRBase by providing higher automation and accuracy for miRNA classification. When measuring sequence similarity, unlike BLAST  or other BLAST-based methods, miRFam uses shorter sequence segments, thus it has a much smaller search space, which allows it to run faster. As the first miRNA-oriented sncRNA family classification method, miRFam has several advantages: (1) Only primary information of miRNAs is required, no other assumption (e.g., common secondary structures within a family or limitation of sequence length) is imposed. (2) Compared with multiple sequence alignment (MSA), miRFam is more efficient and accurate. To classify ~10,000 pre-miRNA sequences, MSA will cost several hours while miRFam consumes only several minutes. (3) miRFam is insensitive to sequencing error and the exact position of pre-miRNA in pri-miRNA. The change of several bases has very little effect on the feature vectors.
Single family tests
Synthetic dataset test
Results of single family experiments
Real dataset test
Results of different combinations of n-gram types
Acc(tri-, bi-&unigram) a
Acc(tri-, bi-&unigram) b
The effect of concentration factor
In this paper, we introduced the concentration factor to weight the features of family vectors (see Equ. 2). Intuitively, the longer fragments of sequences should be more informative than the shorter ones. For example, with some exceptions , a triplet codon in a nucleic acid sequence specifies a single amino acid. And here, a trigram is exactly a triplet. Thus, in representing miRNAs sequences, the longer n-grams should outweigh the shorter ones. In what follows, we will see whether our concentration factor weighting scheme conforms to the above intuition and observation, by checking the centers (before and after weighting) of the three families (let-7, mir-17 and mir-9) and dataset S (the mixed snoRNA class).
In summary, the analysis on the feature vectors of different families shows that the concentration factor weighting scheme can enhance the trigrams while restraining the bigrams and unigrams, which is reasonable and consistent to the intuition and observation. Most importantly, our extensive classification experiments in this and the later sections also show indirectly that the weighting scheme is effective.
As mentioned before, with the development of powerful deep sequencing technology, more miRNA genes will be identified in the future. But the number of real miRNAs in a certain genome is still unknown. Thus, a major concern is how well miRFam will perform if only a small number of known miRNAs are available for some certain families and species. In the previous single family tests, we have employed three types of n-grams (unigrams, bigrams and trigrams) as features, so one natural question is how the different combinations of these types of n-grams will impact miRFam's performance. Furthermore, as mature miRNAs and hairpin sequences are somehow a little different, it occurs to us whether miRFam will perform differently on them. To answer these questions, we tested miRFam on three multi-family datasets constructed from miRBase (version 14) according to their family members. T20 contains the top 20 biggest families in miRBase (version 14), while G1 and G2 contain those families whose members are around 40 and 20, respectively. Here, the numbers 40 and 20 are randomly selected. Performance measurements like sensitivity and specificity are usually defined for binary classification. Here we actually deal with multi-class (i.e. multi-family) classification, so we use accuracy (Acc) as the performance indicator.
The impact of training dataset size
The impact of the combination of different n-gram types
Here, we examine how classification performance will be impacted by the different combinations of unigrams, bigrams and trigrams on these multi-family datasets (Table 2). Actually, we also test miRFam with tetragrams, the results are presented in Additional file 1, Table S3.
We found that miRFam performs better when more types of n-gram features were used. Even when only the trigrams were used to classify miRNAs, the accuracy is around 90%. For the G2 dataset, when features of unigram, bigram and trigram types were all included, the accuracy was surprisedly more than 99%. Further exploring the classification results, we also found that some abnormal sequences with noise bases (not A, U, G and C) were also classified correctly in 5-fold cross validation (sequences are listed in Table S2 in Additional file 1), which means that miRFam is insensitive to base changes, such as single-nucleotide polymorphism (SNP) or sequencing error.
In addition, by transforming pre-miRNA sequences to feature vectors, both normal and abnormal sequences were handled in a similar process, thus avoiding the cumbersome addition, deletion and modification operations used in MSA.
Test with mature miRNAs
Results on mature miRNAs
Acc(tri-, bi-&unigram, %)
Application-oriented large-scale families tests
Performance of large-scale miRBase families test
Accuracy (%) c
Accuracy (%) d
Since 5-fold cross validation was employed, families that contain less than 5 members were not considered at first. A detailed family distribution in miRBase could be found in Figure S3 in the additional file 1. From miRBase v14, the 334 families that contain no less than 5 members were selected, which hold 87.49% (7797/8912) pre-miRNA sequences of the whole database. On this dataset, miRFam achieved an accuracy of 98.18%.
When we were preparing this manuscript, miRBase (version 15) was released in April 2010. This is a significant update, with over 3000 new hairpin sequences and more than 4000 new mature sequences. From miRBase v15, 398 families were selected, each of which contains no less than 5 members. These families constitute 84.38% (9379/11115) hairpin sequences in the whole database. Even with such large-scale families, miRFam still got an accuracy of 97.97%.
When dealing with miRBase v15, there are still 1736 pre-miRNAs distributed in 658 families that were not processed (see Figure S3). Among them, 351 families have only 2 members. In the final experiment, we tested miRFam on the whole 1056 families in miRBase v15. For those families with less than 5 members, we randomly chose one member as the testing sample, and the remaining as training samples, miRFam still obtained an accuracy of 90.66%, which was a surprisingly satisfactory result, considering that classifying a dataset with a large number of classes and the extremely uneven distributions of members in these classes is a well-recognized challenging task.
Effectively classifying newly detected miRNAs to their corresponding families is helpful for their further functional analysis. However, only a few works have been done to address this issue, which is far from being established. Unlike existing alignment-based sncRNA clustering or classification methods [23, 33, 34], which can also be used to cluster or classify miRNAs, the proposed miRFam bases on supervised learning techniques, which is more general and effective. It does not require sequence- or structure-based alignment, thus it is free from the difficulty of choosing multiple parameters used in the alignment-based methods, and is also free from the quality issue of miRNA secondary structure prediction. Certainly, miRFam is not completely parameter-free, it still has to set two parameters, i.e., the feature vector length l and the trade-off between training error and margin c. Another advantage of the miRFam method is its efficiency, especially when the number of sequences is huge. Furthermore, miRFam can achieve satisfactory classification performance over the family system prepared by miRBase. Of all predictions made by miRFam, the accuracy is beyond 90%. Therefore, it can be used to replace the manual modification, which will greatly save time.
Most known miRNA sequences are evolutionary conserved , miRNA families may have consensus secondary structures , and the microRNA-target relationships are also conservative . As people's interest in the miRNA world continuously grows, more and more datasets are going to appear. Correspondingly, there is an urgent need to classify the newly discovered miRNAs into their corresponding families according to sequence and/or structure similarities. With correct family classification, it is easier to elucidate the structures and functions of the new sequences, by using multiple sequence alignments. Apparently, more in-depth information can also be available, such as SNPs within pre-miRNAs and mature miRNAs .
One potential limitation of the proposed approach is that it relies on a prepared family classification structure. Actually, this is a common problem with classification - a supervised machine learning approach, and the quality of training sets significantly influences classification accuracy. To overcome this limitation, we can turn to clustering analysis, which is an unsupervised learning approach that can automatically group the miRNA sequences into different categories based on their characteristics of sequences and/or structures. We keep this issue as our future work.
Sequence alignments are useful for the analysis of genomic data. For example, miRNA genes in newly sequenced organism can be detected based on their homology to genes in related and well-studied species [4, 38]. Once homologous genes are detected, one can perform a MSA with the hope of establishing miRNA families. However, MSA is time consuming in doing this work, different MSA algorithms may build quite different alignments, and choosing an appropriate alignment algorithm is crucial to the performance of family classification.
miRFam can effectively distinguish synthetic random sequences and similar snoRNA sequences from real pre-miRNA sequences (Table 1).
Both precursors and mature miRNAs can be used to infer miRNA families. With shorter mature sequences, miRFam can achieve better classification result (Table 3).
When the dataset contains more than 300 families and each family holds no less than 5 members, the classification accuracy is around 98%. Even with the entire miRBase (version 15, 1056 families and more than 650 of them hold less than 5 samples), the accuracy surprisedly reaches 90% (Table 4).
In summary, we proposed the first supervised learning based approach miRFam to automatically assign miRNA precursors to their corresponding families with high accuracy. It can be useful to help family classification, especially in the applications that previously have been done manually, such as miRBase. Additionally, due to its robustness, miRFam can be used in a wide range of scenarios, as long as an existing family assignment information is available. Certainly, its performance depends on the existing family assignment information. However, as there is more and more study on miRNA, it is foreseeable that more miRNAs will be identified and registered in miRBase. Such a situation will certainly favor the existence and utilization of the miRFam method. In return, miRFam will also contribute a lot to the efficient exploration of these newly discovered miRNAs.
Notations of datasets
reverse sequences of the biggest three miRNA families
combination of SNORA26 and SNORA33 from Rfam10.0
20 families with the largest members, ANM b = 109.9
10 families selected from miRBase14, ANM b = 43.8
10 families selected from miRBase14, ANM b = 20.2
We first ranked miRNA families in miRBase according to the number of members contained in each family. R contains three subsets R1, R2 and R3, corresponding to the three biggest families in miRBase v14 (let-7, mir-17 and mir-9). R1, R2 and R3 were constructed by reversing the original pre-miRNA sequences in let-7, mir-17 and mir-9 with squid, respectively. S was constructed by mixing SNORA26 and SNORA33 downloaded from Rfam v10.0.
SNORA26 (RF00568) is a member of the H/ACA class of small nucleolar RNAs, while SNORA33 (RF00133) is a member of the C/D box class. After being filtered to less than 90% identity, they contain 195 and 122 sequences, respectively. Three multi families datasets (T20, G1, G2) were constructed from miRBase v14 based on the result of family ranking. The biggest family in G1 is mir-33 containing 47 members, and the smallest family is mir-26 containing 41 members. While the biggest (smallest) families in G2 is mir-315 (mir-320), containing 21 (20) miRNAs (Additional file 1, Table S1).
In this paper, we treat family establishment as a classification problem. The first step is to transform miRNA sequences to numeric vectors, which are usually called feature vectors. Here, n-grams  are used as features of miRNA sequences.
An n-gram is a subsequence consisting of n spatially consecutive items from a given sequence. The items in this study are pre-miRNA bases (A,C,G and U). A n-gram of size 1 (i.e. n = 1) is referred to as a "unigram", size 2 (n = 2) is a "bigram", size 3 (n = 3) is a "trigram", size 4 is a "tetragram", and size 5 or more (i.e. n ≥ 5) is simply called a "n-gram". In the sequel, we also call unigrams, bigrams, trigrams and tetragram as type 1, 2, 3 and 4 n-grams, and so on. n-grams can be used for efficient approximate matching. By converting a miRNA precursor to a set of n-grams, it can be embedded into a vector space, thus allowing a sequence to be compared with others in an efficient manner. Here, we select unigrams, bigrams, trigrams and tetragram as features.
To extract n-grams, we use a window of size n that slides on pre-miRNA sequences from 5' to 3'. At each position on a sequence, the subsequence of length n covered by the sliding window corresponds to a n-gram. As the window slides forward, the occurrence frequency t of each encountered n-gram is recorded.
Above, t j is the occurrence frequency of a certain unique n-gram of type i, and T i is the total occurrence frequency of all unique n-grams of type i. A feature vector contains 340 dimensions, each of which corresponds to a unique n-gram of a certain type i (i = 1, 2, 3 and 4). Within a vector, the dimensions are arranged in the order of tetragrams, trigrams, bigrams and unigrams. The sum of all dimensional values of a feature vector is 1.
Binary classification using support vector machine (SVM) is a well developed technique. However, due to performance reasons, using a single SVM formulation directly to solve the multiclass problem is usually avoided. A better approach is to use a combination of several binary SVM classifiers to solve the multiclass problem. Typical algorithms of multiclass learning include the multiclass extensions to decision tree learning  and various specialized versions of the boosting approach such as AdaBoost.M2 and AdaBoost.MH [42, 43]. However, the dominate approach to the multiclass problem is multiclass SVM. One of the most widely-used multiclass SVM methods is one-versus-all. In this method, M binary classifiers are constructed. The i-th classifier's output function F i is trained by using the examples from class i as positives and the examples from all other classes as negatives. For a new example x, the one-versus-all SVM strategy assigns it to the class with the largest value of F i .
In this study, we use the popular multiclass SVM package SVM multiclass (version 2.20). SVM multiclass uses the multi-class formulation described in , and is optimized so that it is very fast in linear cases .
MSA implementation and visualization
Here, TP, FP, TN and FN are the numbers of true positive predictions, false positive predictions, true negative predictions and false negative predictions, respectively.
Conflict of interests
The authors declare that they have no competing interests.
This research was supported by Major State Basic Research and Development Program of China (973 Program) under grant no. 2010CB126604. JG was also supported by the Open Research Program of Shanghai Key Lab of Intelligent Information Processing.
The authors are grateful to Prof. Sam Griffiths-Jones for his useful information about the miRNA family construction in miRBase, and to Prof. Uwe Ohler, Prof. Zhongzhi Zhang and Honglei Ji for their critical suggestion on experiment design. We also thank the authors of squid, SVM multiclass , Clustal W and Jalview who have made their software packages publicly.
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