Full-length analysis of Caenorhabditis elegans transcriptome using Nanopore sequencing technology

A recent meta-analysis of alternative exon usage in Caenorhabditis elegans (Tourasse et al., Genome Research, 2017) refined our comprehension of its transcriptome, especially regarding the splicing quantitative aspects of alternative splicing in messenger RNAs. However, Next-Generation Sequencing technologies like Illumina technology are proving to be limited to fully characterize one’s transcriptome. PCR-based sequencing methods are known to introduce amplification bias affecting the overall distribution of mRNAs detected in one experiment and short-reads are not suited to accurately predict the frequency of isoforms derived from multiple alternative splicing events.

In this study, we are exploiting new possibilities offered by Oxford Nanopore Technology (ONT) to overcome those limitations. Nanopore-based sequencing allow to directly sequence nucleic acids without any prior amplification step and generates long-reads covering up to the full-length of the molecule. Hence, we are aiming to further characterize C. elegans transcriptome by providing a more accurate measure of isoforms ratios, a better comprehension of exons associations during alternative splicing and by characterizing differentially trans-spliced mRNAs.

We sequenced two different populations of mRNAs on a MinION device: a library of poly(A) mRNAs representing the whole-animal transcriptome and a library of SL1-enriched mRNAs. The two libraries were compared together at the level of both genes and isoforms. We are reporting a set of non-SL1 genes that are found highly expressed in poly(A) libraries but not detected in SL1-enriched libraries. Additionally, we are also showing that alternatives promoters can lead to populations of isoforms exhibiting different trans-splicing status.