At the London Calling 2017 conference hosted by Oxford Nanopore Technologies (ONT) Christopher Vollmer’s group (UCSC) showed identification and quantification of transcript isoforms when sequencing amplified cDNA from single cells. Lexogen’s Spike-In RNA Variants (SIRVs) were used here to benchmark the experimental and computation approaches, and a largely unbiased quantification was observed as well as full-length coverage of spike-in transcripts of 0.5 – 2.5 kB length (see details in the group’s bioRxiv manuscript). This application is also documented by ONT in a blog entry on full-length PCR-based and PCR-free cDNA libraries.
Libby Snell of Oxford Nanopore then presented data from direct RNA sequencing (direct RNA-seq, explained in this YouTube video) of yeast RNA and external controls. Lexogen is delighted that its Spike-In RNA Variants (SIRVs) were among the first RNAs to be sequenced directly. Due to their isoform complexity, SIRVs are perfect controls for validating methods that can potentially identify transcript variants with long reads on the RNA level, without the need for cDNA synthesis or short read alignment. However, as this blog entry by ONT reveals, while it was possible to detect and quantify the single-isoform ERCC controls accurately, the high similarity of the SIRV transcripts (6-18 isoforms per SIRV gene) still presents challenges for accurately mapping all individual isoforms in the SIRV mix.
Lexogen is looking forward to seeing this game-changing method developed further to enable accurate identification and quantification of all SIRV isoforms. To aid in the setup of this and all other RNA-seq experiments requiring RNA spike-in controls, Lexogen will release new SIRV mixes. Among the first will be a predefined mix of the 69 SIRV isoforms and the 92 non-variant ERCC transcripts, covering technical validation as well as the more complex issues of isoform identification and concordance of experiments in one easy-to-use mix.