Direct RNA sequencing
Fatih Ozsolak, Adam R. Platt, Dan R. Jones, Jeffrey G. Reifenberger, Lauryn E. Sass, Peter McInerney, John F. Thompson, Jayson Bowers, Mirna Jarosz & Patrice M. Milos
Our understanding of human biology and disease is ultimately dependent on a complete understanding of the genome and its functions. The recent application of microarray and sequencing technologies to transcriptomics has changed the simplistic view of transcriptomes to a more complicated view of genome-wide transcription where a large fraction of transcripts emanates from unannotated parts of genomes1–7, and underlined our limited knowledge of the dynamic state of transcription. Most of this broad body of knowledge was obtained indirectly because current transcriptome analysis methods typically require RNA to be converted to complementary DNA (cDNA) before measurements, even though the cDNA synthesis step introduces multiple biases and artefacts that interfere with both the proper characterization and quantification of transcripts8–18. Furthermore, cDNA synthesis is not particularly suitable for the analysis of short, degraded and/or small quantity RNA samples. Here we report direct single molecule RNA sequencing without prior conversion of RNA to cDNA. We applied this technology to sequence femtomole quantities of poly(A) Saccharomyces cerevisiae RNA using a surface coated with poly(dT) oligonucleotides to capture the RNAs at their natural poly(A) tails and initiate sequencing by synthesis. We observed transcript 39 end heterogeneity and polyadenylated small nucleolar.