Abstract
Long-read sequencing (LRS), a powerful novel approach, is able to read full-length transcripts and confers a major advantage over the earlier gold standard short-read sequencing in the efficiency of identifying for example polycistronic transcripts and transcript isoforms, including transcript length- and splice variants. In this work, we profile the human cytomegalovirus transcriptome using two third-generation LRS platforms: the Sequel from Pacific BioSciences, and MinION from Oxford Nanopore Technologies. We carried out both cDNA and direct RNA sequencing, and applied the LoRTIA software, developed in our laboratory, for the transcript annotations. This study identified a large number of novel transcript variants, including splice isoforms and transcript start and end site isoforms, as well as putative mRNAs with truncated in-frame ORFs (located within the larger ORFs of the canonical mRNAs), which potentially encode N-terminally truncated polypeptides. Our work also disclosed a highly complex meshwork of transcriptional read-throughs and overlaps.
Highlights
Long-read sequencing (LRS), a powerful novel approach, is able to read full-length transcripts and confers a major advantage over the earlier gold standard short-read sequencing in the efficiency of identifying for example polycistronic transcripts and transcript isoforms, including transcript length- and splice variants
As dRNA-Seq is free of reverse transcription (RT)- and PCR-biases, it can be used for the validation of introns
Due to its lower coverage and shorter average read lengths (Table 1 and Fig. 1) compared to the cDNA libraries, and its other biases, dRNA-Seq is advised to use in conjunction with other methods
Summary
Long-read sequencing (LRS), a powerful novel approach, is able to read full-length transcripts and confers a major advantage over the earlier gold standard short-read sequencing in the efficiency of identifying for example polycistronic transcripts and transcript isoforms, including transcript length- and splice variants. We profile the human cytomegalovirus transcriptome using two third-generation LRS platforms: the Sequel from Pacific BioSciences, and MinION from Oxford Nanopore Technologies We carried out both cDNA and direct RNA sequencing, and applied the LoRTIA software, developed in our laboratory, for the transcript annotations. LRS platforms are currently commercially available by Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT), which provide read lengths of ∼15 kb for PacBio and > 30 kb for ONT that surpass lengths of most transcripts Both techniques were applied for the investigation of transcriptomic complexity of human cell lines[7] and various organisms, such as mammals8, fish[9] and p lants[10] and a number of viruses, such as poxviruses11, baculoviruses12, coronaviruses13, circoviruses14, adenoviruses[15]; and herpesviruses[16,17,18,19]. Short-read RNA sequencing studies have discovered splice junctions and n cRNAs39 and have shown that the most abundant HCMV transcripts are expressed in different cell types[10]
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