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BMC Genomics

, 17:220

Transcriptomic methods


BackgroundRecent advances in sequencing technology have opened a new era in RNA studies. Novel types of RNAs such as long non-coding RNAs lncRNAs have been discovered by transcriptomic sequencing and some lncRNAs have been found to play essential roles in biological processes. However, only limited information is available for lncRNAs in Drosophila melanogaster, an important model organism. Therefore, the characterization of lncRNAs and identification of new lncRNAs in D. melanogaster is an important area of research. Moreover, there is an increasing interest in the use of ChIP-seq data H3K4me3, H3K36me3 and Pol II to detect signatures of active transcription for reported lncRNAs.

ResultsWe have developed a computational approach to identify new lncRNAs from two tissue-specific RNA-seq datasets using the polyA-enriched and the ribo-zero method, respectively. In our results, we identified 462 novel lncRNA transcripts, which we combined with 4137 previously published lncRNA transcripts into a curated dataset. We then utilized 61 RNA-seq and 32 ChIP-seq datasets to improve the annotation of the curated lncRNAs with regards to transcriptional direction, exon regions, classification, expression in the brain, possession of a polyA tail, and presence of conventional chromatin signatures. Furthermore, we used 30 time-course RNA-seq datasets and 32 ChIP-seq datasets to investigate whether the lncRNAs reported by RNA-seq have active transcription signatures. The results showed that more than half of the reported lncRNAs did not have chromatin signatures related to active transcription. To clarify this issue, we conducted RT-qPCR experiments and found that ~95.24 % of the selected lncRNAs were truly transcribed, regardless of whether they were associated with active chromatin signatures or not.

ConclusionsIn this study, we discovered a large number of novel lncRNAs, which suggests that many remain to be identified in D. melanogaster. For the lncRNAs that are known, we improved their characterization by integrating a large number of sequencing datasets 93 sets in total from multiple sources lncRNAs, RNA-seq and ChIP-seq. The RT-qPCR experiments demonstrated that RNA-seq is a reliable platform to discover lncRNAs. This set of curated lncRNAs with improved annotations can serve as an important resource for investigating the function of lncRNAs in D. melanogaster.

KeywordsLong non-coding RNA Active transcription ChIP-seq RNA-seq Drosophila melanogaster AbbreviationsChIP-seqChromatin immunoprecipitation sequencing

CPCCoding potential calculator

H3K4me3Tri-methylation of H3 lysine 4

H3K36me3Tri-methylation of H3 lysine 36

lincRNALong intergenic non-coding RNA

lncRNALong non-coding RNA

MSLMale specific lethal

Pol IIRNA polymerase II

RT-qPCRQuantitative reverse transcriptase-dependent polymerase chain reaction

TSStranscription start site

Mei-Ju May Chen and Li-Kai Chen as co-first authors.

Electronic supplementary materialThe online version of this article doi:10.1186-s12864-016-2457-0 contains supplementary material, which is available to authorized users.

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Autor: Mei-Ju May Chen - Li-Kai Chen - Yu-Shing Lai - Yu-Yu Lin - Dung-Chi Wu - Yi-An Tung - Kwei-Yan Liu - Hsueh-Tzu Shih - Yi-


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