Abstract
A platform for highly parallel direct sequencing of native RNA strands was recently described by Oxford Nanopore Technologies, but despite initial efforts it remains crucial to further investigate the technology for quantification of complex transcriptomes. Here we undertake native RNA sequencing of polyA + RNA from two human cell lines, analysing ~5.2 million aligned native RNA reads. To enable informative comparisons, we also perform relevant ONT direct cDNA- and Illumina-sequencing. We find that while native RNA sequencing does enable some of the anticipated advantages, key unexpected aspects currently hamper its performance, most notably the quite frequent inability to obtain full-length transcripts from single reads, as well as difficulties to unambiguously infer their true transcript of origin. While characterising issues that need to be addressed when investigating more complex transcriptomes, our study highlights that with some defined improvements, native RNA sequencing could be an important addition to the mammalian transcriptomics toolbox.
Highlights
A platform for highly parallel direct sequencing of native RNA strands was recently described by Oxford Nanopore Technologies, but despite initial efforts it remains crucial to further investigate the technology for quantification of complex transcriptomes
We observed that despite the fact that Oxford Nanopore Technologies (ONT) reads are around an order of magnitude longer than typical Illumina reads, identification of their transcript of origin is still highly nontrivial, and a large number of secondary transcriptome alignments with mapping scores very close to the primary alignments were observed for all libraries
We expect that referencebased transcript abundance estimation methods that are able to incorporate information about these multimapping reads are more likely to produce reliable abundance estimates; to our knowledge no such ONT-specific method, with a read generation model adapted to the ONT data characteristics, currently exists
Summary
A platform for highly parallel direct sequencing of native RNA strands was recently described by Oxford Nanopore Technologies, but despite initial efforts it remains crucial to further investigate the technology for quantification of complex transcriptomes. The ability to readily perform transcript-level functional investigations will almost certainly enrich our understanding of a number of important biological processes To enable this to be accomplished in a reliable manner, methods that can unequivocally distinguish and quantify the presence of transcript isoforms from raw sequence reads are required. Long-read sequencing methodologies have been introduced into the transcriptomics field, offering the opportunity to directly generate individual reads that can span the full length of transcripts[6–12]. We apply ONT long-read native RNA sequencing to samples from two human cell lines; HAP1 and HEK293, with the primary aim of evaluating the ability to identify and quantify transcripts and genes in a complex transcriptome setting. We present our findings relating to differences between the performance of a variety of analysis algorithms, and the potential advantages that current ONT direct RNA-seq brings over the traditional Illumina sequencing, as well as current limitations of the technology
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