The full-length transcriptome ofC. elegansusing direct RNA sequencing

Abstract

Current transcriptome annotations have largely relied on short read lengths intrinsic to most widely used high-throughput cDNA sequencing technologies. For example, in the annotation of the Caenorhabditis elegans transcriptome, more than half of the transcript isoforms lack full-length support and instead rely on inference from short reads that do not span the full length of the isoform. We applied nanopore-based direct RNA sequencing to characterize the developmental polyadenylated transcriptome of C. elegans . Taking advantage of long reads spanning the full length of mRNA transcripts, we provide support for 20,902 splice isoforms across 14,115 genes, without the need for computational reconstruction of gene models. Of the isoforms identified, 2,188 are novel splice isoforms not present in the Wormbase WS265 annotation. Furthermore, we identified 16,325 3' untranslated region (3'UTR) isoforms, 2,304 of which are novel and do not fall within 10 bp of existing 3'UTR datasets and annotations. Combining 3'UTRs and splice isoforms we identified 25,944 full-length isoforms. We also determined that poly(A) tail lengths of transcripts vary across development, as do the strengths of previously reported correlations between poly(A) tail length and expression level, and poly(A) tail length and 3'UTR length. Finally, we have formatted this data as a publically accessible track hub, enabling researchers to explore this dataset easily in a genome browser.

Quantitative profiling of pseudouridylation dynamics in native RNAs with nanopore sequencing

“…Direct RNA nanopore sequencing has been successfully applied in a wide variety of organisms [29][30][31][84][85][86][87] . However, the detection of distinct RNA modification types in individual native RNA molecules is still an unsolved challenge.…”

Innate Immunity in the C. elegans Intestine Is Programmed by a Neuronal Regulator of AWC Olfactory Neuron Development

“…The resulting sam files were converted to bam format using samtools view with parameters: -b -F 2048 . Read filtering and splice isoform identification were analyzed as described (Roach et al, 2019). The GTF (WS220/ce11) annotation file was downloaded from UCSC genome website and the assembled GTF file was generated for each sample using Stringtie (version 1.3.4).…”

Evolution of Yin and Yang isoforms of a chromatin remodeling subunit precedes the creation of two genes

“…To identify other transcripts produced by nurf-1 and quantify the relative proportions of each that are produced, we analyzed previously published Illumina short-read (Brunquell et al, 2016) (isolated from synchronized L2 larval animals) and Oxford Nanopore long-read RNA sequencing reads (Roach et al, 2019) (isolated from mixed populations) (Figure 2—figure supplements 1–2). Our results support many of the conclusions of Andersen et al (2006) but contain a few surprises.…”