Identification of Non-coding RNAs by High-Throughput Sequencing

Non-coding RNA transcripts, such as long non-coding RNAs, miRNAs, siRNAs, and transposon-originating transcripts, are involved in the regulation of RNA stability, protein translation, and/or the modulation of chromatin states. RNA-Seq can be used to catalog this diversity of novel transcripts and a joint analysis of these transcriptomic data can provide useful insights into epigenetic regulation of dynamic responses such as the stress response, which may not be deciphered from individual analysis of single transcript categories. Here, researchers from the University of Padova present a protocol that allows the identification and analysis of small RNAs and long non-coding RNAs, together with the comparison of these species between different sample types.

Genome browser view of RNA-Seq and sRNA-Seqreads mapped at
Class U and Class X newly identified loci

rna-seq

IGV—Integrative Genomics Viewer (http://software.broadinstitute.org/software/igv/) snapshots reporting mapped reads, annotated transcript (blue boxes) and repeat annotations (black boxes) for three maize genome regions in which new transcripts have been annotated. (a) Coverage of RNA directional sequencing reads (normalized to the total of mapped reads) for B73 (blue) and rpd1/rmr6 mutant (red) showing a rpd1/rmr6 30 Kb transcriptional active region on chromosome 5 with several newly annotated Class U intergenic loci. siRNAs are particularly abundant at the boundaries of this region in which several RLG class I transposable elements are annotated. (b) Strand-specific coverage and mapped reads at a newly identified Class X locus, transcribed in both B73 and rpd1/rmr6 on the opposite strand of the AC216891_FGT004 transcript. Antisense AC216891_FGT004_X_1 corresponds to an RLC class I transposable element, which is completely covered by siRNAs. (c) Strand-specific coverage and mapped reads on a chromosome 8 complex region. Blue mapped reads indicate transcription of the plus strand in both B73 and rpd1/rmr6, while red reads, mapped on the minus strand, are detectable only in the rpd1/rmr6 mutant, resulting in the annotation of both antisense and intergenic new loci. Several peaks of small RNA reads are detectable in this region

Lunardon A, Forestan C, Farinati S, Varotto S. (2017) De Novo Identification of sRNA Loci and Non-coding RNAs by High-Throughput Sequencing. Methods Mol Biol 1675:297-314. [abstract]

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