Review Article Uncovering the Complexity of Transcriptomes with RNA-Seq

Hindawi Publishing Corporation Journal of Biomedicine and Biotechnology Volume 2010, Article ID 853916, 19 pages doi:10.1155/2010/853916 Review Article Uncovering the Complexity of Transcriptomes with RNA-Seq Valerio Costa,1 Claudia Angelini,2 Italia De Feis,2 and Alfredo Ciccodicola1 1 Institute of Genetics and Biophysics “A. Buzzati-Traverso”, IGB-CNR, 80131 Naples, Italy 2 Istituto per le Applicazioni del Calcolo “Mauro Picone”, IAC-CNR, 80131 Naples, Italy Correspondence should be addressed to Valerio Costa, [email protected] Received 22 February 2010; Accepted 7 April 2010 Academic Editor: Momiao Xiong Copyright © 2010 Valerio Costa et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In recent years, the introduction of massively parallel sequencing platforms for Next Generation Sequencing (NGS) protocols, able to simultaneously sequence hundred thousand DNA fragments, dramatically changed the landscape of the genetics studies. RNA-Seq for transcriptome studies, Chip-Seq for DNA-proteins interaction, CNV-Seq for large genome nucleotide variations are only some of the intriguing new applications supported by these innovative platforms. Among them RNA-Seq is perhaps the most complex NGS application. Expression levels of specific genes, differential splicing, allele-specific expression of transcripts can be accurately determined by RNA-Seq experiments to address many biological-related issues. All these attributes are not readily achievable from previously widespread hybridization-based or tag sequence-based approaches. However, the unprecedented level of sensitivity and the large amount of available data produced by NGS platforms provide clear advantages as well as new challenges and issues. This technology brings the great power to make several new biological observations and discoveries, it also requires a considerable effort in the development of new bioinformatics tools to deal with these massive data files. The paper aims to give a survey of the RNA-Seq methodology, particularly focusing on the challenges that this application presents both from a biological and a bioinformatics point of view. 1. Introduction ribosomal RNA (80–90%, rRNA), transfer RNA (5–15%, tRNA), mRNA (2–4%) and a small fraction of intragenic It is commonly known that the genetic information is (i.e., intronic) and intergenic noncoding RNA (1%, ncRNA) conveyed from DNA to proteins via the messenger RNA with undefined regulatory functions [3]. Particularly, both (mRNA) through a finely regulated process. To achieve intragenic and intergenic sequences, enriched in repetitive such a regulation, the concerted action of multiple cis- elements, have long been considered genetically inert, mainly acting proteins that bind to gene flanking regions—“core” composed of “junk” or “selfish” DNA [4]. More recently and “auxiliary” regions—is necessary [1]. In particular, it has been shown that the amount of noncoding DNA core elements, located at the exons’ boundaries, are strictly (ncDNA) increases with organism complexity, ranging from required for initiating the pre-mRNA processing events, 0.25% of prokaryotes’ genome to 98.8% of humans [5]. whereas auxiliary elements, variable in number and location, These observations have strengthened the evidence that are crucial for their ability to enhance or inhibit the basal ncDNA, rather than being junk DNA, is likely to represent splicing activity of a gene. the main driving force accounting for diversity and biological Until recently—less than 10 years ago—the central complexity of living organisms. dogma of genetics indicated with the term “gene” a DNA Since the dawn of genetics, the relationship between portion whose corresponding mRNA encodes a protein. DNA content and biological complexity of living organisms According to this view, RNA was considered a “bridge” in has been a fruitful field of speculation and debate [6]. To the transfer of biological information between DNA and date, several studies, including recent analyses performed proteins, whereas the identity of each expressed gene, and during the ENCODE project, have shown the pervasive of its transcriptional levels, were commonly indicated as nature of eukaryotic transcription with almost the full length “transcriptome” [2]. It was considered to mainly consist of of nonrepeat regions of the genome being transcribed [7]. 2 Journal of Biomedicine and Biotechnology The unexpected level of complexity emerging with the reads in excess of 400 bp. It was followed in 2006 by discovery of endogenous small interfering RNA (siRNA) and the Illumina Genome Analyzer (GA) (http://www.illumina microRNA (miRNA) was only the tip of the iceberg [8]. .com/) capable to generate tens of millions of 32-bp reads. Long interspersed noncoding RNA (lincRNA), promoter- Today, the Illumina GAIIx produces 200 million 75–100 bp and terminator-associated small RNA (PASR and TASR, reads. The last to arrive in the marketplace was the Applied resp.), transcription start site-associated RNA (TSSa-RNA), Biosystems platform based on Sequencing by Oligo Ligation transcription initiation RNA (tiRNA) and many others [8] and Detection (SOLiD) (http://www3.appliedbiosystems represent part of the interspersed and crosslinking pieces .com/AB Home/index.htm), capable of producing 400 mil- of a complicated transcription puzzle. Moreover, to cause lion 50-bp reads, and the Helicos BioScience HeliS- further difficulties, there is the evidence that most of the cope (http://www.helicosbio.com/), the first single-molecule pervasive transcripts identified thus far, have been found sequencer that produces 400 millions 25–35 bp reads. only in specific cell lines (in most of cases in mutant cell lines) While the individual approaches considerably vary in with particular growth conditions, and/or particular tissues. their technical details, the essence of these systems is the In light of this, discovering and interpreting the complexity miniaturization of individual sequencing reactions. Each of of a transcriptome represents a crucial aim for understanding these miniaturized reactions is seeded with DNA molecules, the functional elements of such a genome. Revealing the at limiting dilutions, such that there is a single DNA molecule complexity of the genetic code of living organisms by in each, which is first amplified and then sequenced. To be analyzing the molecular constituents of cells and tissues, will more precise, the genomic DNA is randomly broken into drive towards a more complete knowledge of many biological smaller sizes from which either fragment templates or mate- issues such as the onset of disease and progression. pair templates are created. A common theme among NGS The main goal of the whole transcriptome analyses is technologies is that the template is attached to a solid surface to identify, characterize and catalogue all the transcripts or support (immobilization by primer or template) or indi- expressed within a specific cell/tissue—at a particular stage— rectly immobilized (by linking a polymerase to the support). with the great potential to determine the correct splicing The immobilization of spatially separated templates allows patterns and the structure of genes, and to quantify the simultaneous thousands to billions of sequencing reactions. differential expression of transcripts in both physio- and The physical design of these instruments allows for an pathological conditions [9]. optimal spatial arrangement of each reaction, enabling an In the last 15 years, the development of the hybridiza- efficient readout by laser scanning (or other methods) for tion technology, together with the tag sequence-based millions of individual sequencing reactions onto a standard approaches, allowed to get a first deep insight into this glass slide. While the immense volume of data generated is field, but, beyond a shadow of doubt, the arrival on the attractive, it is arguable that the elimination of the cloning marketplace of the NGS platforms, with all their “Seq” appli- step for the DNA fragments to sequence is the greatest benefit cations, has completely revolutionized the way of thinking of these new technologies. All current methods allow the the molecular biology. direct use of small DNA/RNA fragments not requiring their The aim of this paper is to give an overview of the insertion into a plasmid or other vector, thereby removing RNA-Seq methodology, trying to highlight all the challenges a costly and time-consuming step of traditional Sanger that this application presents from both the biological and sequencing. bioinformatics point of view. It is beyond a shadow of doubt that the arrival of NGS technologies in the marketplace has changed the way we think about scientific approaches in basic, applied and 2. Next Generation Sequencing Technologies clinical research. The broadest application of NGS may be the resequencing of different genomes and in particular, human Since the first complete nucleotide sequence of a gene, pub- genomes to enhance our understanding of how genetic lished in 1964 by Holley [10] and the initial developments differences affect health and disease. Indeed, these platforms of Maxam and Gilbert [11] and Sanger et al. [12] in the have been quickly applied to many genomic contexts giving 1970s (see Figure 1), the world of nucleic acid sequencing rise to the following “Seq” protocols: RNA-Seq for transcrip- was a RNA world and the

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