The full-length transcriptome of C. elegans using direct RNA sequencing

Abstract

Current transcriptome annotations have largely relied on short read lengths intrinsic to the 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 23,865 splice isoforms across 14,611 genes, without the need for computational reconstruction of gene models. Of the isoforms identified, 3452 are novel splice isoforms not present in the WormBase WS265 annotation. Furthermore, we identified 16,342 isoforms in the 3 ′ untranslated region (3 ′ UTR), 2640 of which are novel and do not fall within 10 bp of existing 3 ′-UTR data sets and annotations. Combining 3 ′ UTRs and splice isoforms, we identified 28,858 fulllength transcript 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 publicly accessible track hub, enabling researchers to explore this data set easily in a genome browser.

Nanopore direct RNA sequencing detects differential expression between human cell populations

“…dRNA doesn't utilise any amplification or fragmentation steps and has the potential to quantify both genes and isoforms in an unbiased manner, while also characterising the RNA modifications and polyA tail on each RNA. Studies to date have used dRNA sequencing to catalogue known and novel transcripts in yeast (Jenjaroenpun et al 2018), C.elegans (Roach et al 2020;Li et al 2020), Arabidopsis (Zhang et al 2020) and human cell lines (Workman et al 2019;Soneson et al 2019); characterise polyA tail lengths of individual transcripts (Roach et al 2020;Workman et al 2019); identify allele specific gene and isoform expression (Workman et al 2019); identify RNA base modifications (Workman et al 2019;Garalde et al 2018;Liu et al 2019;Lorenz et al 2019) and infer RNA structure (Stephenson et al 2020). Maximum read lengths for dRNA were also much longer than for PCR-based long-read cDNA sequencing (Workman et al 2019), demonstrating its potential to sequence long and complex RNA splice isoforms.…”

Long-read RNA sequencing of human and animal filarial parasites improves gene models and discovers operons

“…We found the median poly(A) tail length for CCSs from B. malayi was 33 nt (range of 1-541) and 35 (range of 1-536) for D. immitis (Fig. 5A), shorter than the reported C. elegans median of around ~50 nt (depending on life stage and technique) and much shorter than the typical lengths for human organoids, iPSCs, and cell lines [55][56][57] . Nearly 40% of B. malayi…”

Beyond mass spectrometry, the next step in proteomics

“…However, recent developments in nanopore sequencing have allowed for direct sequencing of RNA molecules (94)(95)(96). In particular, long-read sequencing with a nanopore allows for easy resolution of the transcripts without reconstruction; our work (65,66) and others (68,92) suggest that there are many unannotated isoforms even in organisms that have been thoroughly analyzed.…”

“…Here, we offer a less than comprehensive survey (62-64) of five alternatives with the brightest prospects to displace MS in identifying whole proteins and sequencing them. This review starts with schemes that repurpose state-of-the-art, high-throughput, long-read DNA sequencing for transcriptomics to analyze the true mRNA before translation, including the possible isoforms (65)(66)(67)(68)(69). It is a small step from there to cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) (70), which uses oligonucleotide-labeled antibodies to integrate protein and transcriptome measurements together for an efficient readout.…”