Power sequencing

Abstract

Advances in DNA-sequencing technology provide unprecedented insight into the entire collection of four genomes' transcribed sequences; they herald a new era in the study of gene regulation and genome function.Genomes are the blueprints of life: they contain all the information necessary to build and operate their hosts. But we still have much to learn about the language of DNA to interpret the billions of the Gs, As, Ts and Cs, the DNA bases that spell out life. The informationcontaining portions of genomes are transcribed into two RNA classes: messenger RNAs, which are translated into proteins; and non-coding RNAs, which have regulatory, structural or mechanical roles. So studying the transcribed portion of the genome -the transcriptome -significantly aids gene identification, as well as providing insight into the inner workings of the genome and the biology of an organism. Five papers 1-5 , including one on page XXX of this issue by Wilhelm et al. 1 , describe how advances in DNA-sequencing technology can be harnessed to explore transcriptomes in remarkable detail.The concept of sequencing large numbers of randomly selected mRNAs is not new. It forms the basis of the controversial, yet revolutionary EST method 6 , which was originally used to identify genes in the reference copy of the human genome. In this technique, genes are quickly identified through sequencing small fragments of large numbers of mRNAs. Although EST sequencing remains useful, it is relatively slow, requires considerable resources and generally cannot identify mRNAs that are expressed at low levels.DNA microarrays are also powerful tools for transcriptome analysis. Particularly informative are tiling arrays, which are dotted with DNA sequences derived from defined intervals (for example, every 35 base pairs) throughout the genome. Fluorescently labelled RNA is then allowed to bind to the arrays, and the transcribed portions of the genome are identified by determining which DNA sequences pair with the RNA. But tiling arrays also have several shortcomings. First, they can only be used for organisms with known genome sequences. Second, their limited sensitivity, specificity and dynamic range -the ratio between the smallest and largest fluorescent signal -make it difficult to identify lowabundance mRNAs and to distinguish between highly similar mRNA sequences. Finally, the number of DNA probes that fit on a microarray is limited, putting constrains on the

Regulation of circRNA biogenesis

“…13,14 Recently, with the depletion of both polyadenylated RNAs and rRNAs, non-polyadenylated human RNAs could be enriched for RNA sequencing (RNAseq) analysis and unexpected RNA-seq signals were accumulated in certain exons (called excised exons) or introns (called excised introns). 15 These non-polyadenylated signals were further confirmed as circular RNAs from back-spliced exons (circRNAs) 16 or stabilized introns, having escaped debranching (circular intronic RNAs, ciRNAs).…”

Identification of Functional Elements and Regulatory Circuits by Drosophila modENCODE

“…To comprehensively characterize transcribed sequences, we performed RNA-seq using poly(A) + and total RNA, cap analysis of gene expression, rapid amplification of cDNA ends, and produced expressed sequence tags (table S1) (14–16) and cDNAs. These data support more than 90% of annotated genes, exons, and splice junctions and provide experimental evidence for a total of 17,000 protein-coding and noncoding genes, of which 1938 are previously unannotated.…”

The use of high-throughput sequencing methods for plant microRNA research

“…Finally, the short double-stranded cDNA fragments with ligated adapters are sent for sequencing. 19,22 The sequenced reads could be assembled to discover novel transcripts, or mapped to the already known templates for expression level quantification of specific genes.…”