Since transcriptome analysis provides genome-wide sequence and gene expression information, transcript reconstruction using RNA-Seq sequence reads has become popular during recent years. For non-model organism, as distinct from the reference genome-based mapping, sequence reads are processed via de novo transcriptome assembly approaches to produce large numbers of contigs corresponding to coding or non-coding, but expressed, part of genome. In spite of immense potential of RNA-Seq-based methods, particularly in recovering full-length transcripts and spliced isoforms from short-reads, the accurate results can be only obtained by the procedures to be taken in a step-by-step manner. In this chapter, we aim to provide an overview of the state-of-the-art methods including (i) quality check and pre-processing of raw reads, (ii) the pros and cons of de novo transcriptome assemblers, (iii) generating non-redundant transcript data, (iv) current quality assessment tools for de novo transcriptome assemblies, (v) approaches for transcript abundance and diferential expression estimations and inally (vi) further mining of transcriptomic data for particular biological questions. Our intention is to provide an overview and practical guidance for choosing the appropriate approaches to best meet the needs of researchers in this area and also outline the strategies to improve on-going projects.

RNA sequencing (RNA-Seq) is a powerful technique for the gene-expression profiling of organisms that uses the capabilities of next-generation sequencing technologies. Developing gene-expression-based classification algorithms is an emerging powerful method for diagnosis, disease classification and monitoring at molecular level, as well as providing potential markers of diseases. Most of the statistical methods proposed for the classification of geneexpression data are either based on a continuous scale (eg. microarray data) or require a normal distribution assumption. Hence, these methods cannot be directly applied to RNASeq data since they violate both data structure and distributional assumptions. However, it is possible to apply these algorithms with appropriate modifications to RNA-Seq data. One way is to develop count-based classifiers, such as Poisson linear discriminant analysis and negative binomial linear discriminant analysis. Another way is to bring the data closer to microarrays and apply microarray-based classifiers. In this study, we compared several classifiers including PLDA with and without power transformation, NBLDA, single SVM, bagging SVM (bagSVM), classification and regression trees (CART), and random forests (RF). We also examined the effect of several parameters such as overdispersion, sample size, number of genes, number of classes, differential-expression rate, and the transformation method on model performances. A comprehensive simulation study is conducted and the results are compared with the results of two miRNA and two mRNA experimental datasets. The results revealed that increasing the sample size, differential-expression rate and decreasing the dispersion parameter and number of groups lead to an increase in classification accuracy. Similar with differential-expression studies, the classification of RNA-Seq data requires careful attention when handling data overdispersion. We conclude that, as a count-based classifier, the power transformed PLDA and, as a microarray-based classifier, vst or rlog transformed RF and SVM classifiers may be a good choice for classification. An R/BIOCONDUCTOR package, MLSeq, is freely available at https://www.bioconductor.org/packages/release/bioc/ html/MLSeq.html.

Background

No abstract

Background RNA sequencing (RNA-Seq) is a powerful technique for transcriptome profiling of the organisms that uses the capabilities of next-generation sequencing (NGS) technologies. Recent advances in NGS let to measure the expression levels of tens to thousands of transcripts simultaneously. Using such information, developing expression-based classification algorithms is an emerging powerful method for diagnosis, disease classification and monitoring at molecular level, as well as providing potential markers of disease. Microarray based classifiers cannot be directly applied due to the discrete nature of RNA-Seq data. One way is to develop count-based classifiers, such as poisson linear discriminant analysis (PLDA) and negative binomial linear discriminant analysis (NBLDA). Other way is to transform the data hierarchically closer to microarrays and apply microarray-based classifiers. In most of the studies, the data overdispersion seems to be an another challenge in modeling RNA-Seq data. In this study, we aimed to examine the effect of dispersion parameter and classification algorithms on RNA-Seq classification. We also considered the effect of other parameters (i) sample size, (ii) number of genes, (iii) number of class, (iv) DE (differential expression) rate, (v) transformation method on classification performance.