![]() Because Illumina reads are short, they are unlikely to overlap multiple splice junctions, meaning that phasing of splicing events is difficult and requires complex computational reconstruction. ![]() However, when the transcript models are unknown, for example in a non-model organism or a mutant or disease with altered RNA processing, new transcript models must be generated, either de novo or with the aid of the reference genome. Methods exist for quantifying known alternative splicing events from short reads. Illumina RNAseq can generate hundreds of millions of highly accurate short sequencing reads, each representing a 50–250-nt fragment of full-length RNA. Three current technologies use RT-based RNA sequencing library preparation: Illumina, Pacific Biosciences (PacBio), and Oxford Nanopore Technologies (ONT). However, template strand switching by reverse transcriptase (RT) during the copying process can produce spurious splicing patterns and antisense RNA signals. High-throughput sequencing of RNA rarely involves direct RNA sequencing (DRS): instead, copies of complementary DNA (cDNA) produced by reverse transcription of RNA molecules are sequenced. The success of this approach depends upon sequencing methodology and the computational analyses used in interpreting the sequence data. The sequencing of RNAs (RNAseq) can reveal gene expression patterns in specific cells, tissues, or whole organisms. Consequently, the identification and quantification of different RNA processing events is crucial to understand not only what genomes encode but also the biology of whole organisms. Changes in RNA processing can reflect the reprogramming of gene expression patterns during development or in response to stress, or result from genetic mutation or disease. For example, more than 90% of human protein-coding genes have at least two splice isoforms. Patterns of alternative processing can be extensive. Alternative processing choices include distinct transcription start sites, the alternative splicing of different intron and exon combinations, alternative sites of cleavage and polyadenylation, and base modifications such as methylation of adenosines. Eukaryotic transcription by DNA-dependent RNA polymerase II is associated with multiple alternative RNA processing events that diversify the coding and regulatory potential of the genome. Understanding eukaryotic genomes requires knowing not only the DNA sequence but also which RNAs are transcribed from it.
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