reads corresponding to each object is counted. Initially, transcriptomes were analyzed and studied using expressed sequence tags libraries and serial and cap analysis of gene expression (SAGE). Currently, the two main transcriptomics techniques include DNA microarrays and RNA-Seq. Both techniques require RNA isolation through RNA extraction techniques, followed by its separation from other cellular components and enrichment of mRNA. There are two general methods of inferring transcriptome sequences. One approach maps sequence reads onto a reference genome, either of the organism itself (whose transcriptome is being studied) or of a closely related species. The other approach, de novo transcriptome assembly, uses software to infer transcripts directly from short sequence reads and is used in organisms with genomes that are not sequenced. DNA microarrays The first transcriptome studies were based on microarray techniques (also known as DNA chips). Microarrays consist of thin glass layers with spots on which oligonucleotides, known as "probes" are arrayed; each spot contains a known DNA sequence. When performing microarray analyses, mRNA is collected from a control and an experimental sample, the latter usually representative of a disease. The RNA of interest is converted to cDNA to increase its stability and marked with fluorophores of two colors, usually green and red, for the two groups. The cDNA is spread onto the surface of the microarray where it hybridizes with oligonucleotides on the chip and a laser is used to scan. The fluorescence intensity on each spot of the microarray corresponds to the level of gene expression and based on the color of the fluorophores selected, it can be determined which of the samples exhibits higher levels of the mRNA of interest. One microarray usually contains enough oligonucleotides to represent all known genes; however, data obtained using microarrays does not provide information about unknown genes. During the 2010s, microarrays were almost completely replaced by next-generation techniques that are based on DNA sequencing. RNA sequencing RNA sequencing is a next-generation sequencing technology; as such it requires only a small amount of RNA and no previous knowledge of the genome. It allows for both qualitative and quantitative analysis of RNA transcripts, the former allowing discovery of new transcripts and the latter a measure of relative quantities for transcripts in a sample. The three main steps of sequencing transcriptomes of any biological samples include RNA purification, the synthesis of an RNA or cDNA library and sequencing the library. The RNA purification