a result of repeat content in non-coding intron regions of DNA – transcriptome sequence coverage levels can be directly indicative of gene expression levels. These repeated sequences also create ambiguities in the formation of contigs in genome assembly, while ambiguities in transcriptome assembly contigs usually correspond to spliced isoforms, or minor variation among members of a gene family. Genome assembler can't be directly used in transcriptome assembly for several reasons. First, genome sequencing depth is usually the same across a genome, but the depth of transcripts can vary. Second, both strands are always sequenced in genome sequencing, but RNA-seq can be strand-specific. Third, transcriptome assembly is more challenging because transcript variants from the same gene can share exons and are difficult to resolve unambiguously. Method RNA-seq Once RNA is extracted and purified from cells, it is sent to a high-throughput sequencing facility, where it is first reverse transcribed to create a cDNA library. This cDNA can then be fragmented into various lengths depending on the platform used for sequencing. Each of the following platforms utilizes a different type of technology to sequence millions of short reads: 454 Sequencing, Illumina, and SOLiD. Assembly algorithms The cDNA sequence reads are assembled into transcripts via a short read transcript assembly program. Most likely, some amino acid variations among transcripts that are otherwise similar reflect different protein isoforms. It is also possible that they represent different genes within the same gene family, or even genes that share only a conserved domain, depending on the degree of variation. A number of assembly programs are available (see Assemblers). Although these programs have been generally successful in assembling genomes, transcriptome assembly presents some unique challenges. Whereas high sequence coverage for a genome may indicate the presence of repetitive sequences (and thus be masked), for a transcriptome, they may indicate abundance. In addition, unlike genome sequencing, transcriptome sequencing can be strand-specific, due to the possibility of both sense and antisense transcripts. Finally, it can be difficult to reconstruct and tease apart all splicing isoforms. Short read assemblers generally use one of two basic algorithms: overlap graphs and de Bruijn graphs. Overlap graphs are utilized for most assemblers designed for Sanger sequenced reads. The overlaps between each pair of reads is computed and compiled into a graph, in which each node represents a single sequence read. This algorithm is more computationally intensive than de Bruijn graphs, and most effective