Journal Article

Direct RNA sequencing

Thompson, John F., Jarosz, Mirna, Ozsolak, Fatih, Sass, Lauryn E., Reifenberger, Jeffrey G., Jones, Dan R., Platt, Adam R., McInerney, Peter, Milos, Patrice M., Bowers, Jayson

2009

Overview

A direct line to the transcriptome Understanding the functional output of the genome — the sum total of messenger RNAs in a cell or cell population, known as the transcriptome — is an essential step on the way to understanding biology. Current methods for studying the transcriptome rely on microarray and sequencing approaches that require complementary DNA synthesis followed by multiple manipulations, which introduce biases and potential artefacts. Now a team based at Helicos BioSciences Corporation has developed a direct single-molecule RNA sequencing technique that when scaled-up promises a bias-free high-throughput transcriptome analysis. Understanding the functional output of the genome — the transcriptome — is an essential step on the way to understanding human biology and disease. Current transcriptome analysis methods are indirect, typically requiring RNA to be converted to complementary DNA (cDNA) before measurements. Single molecule RNA sequencing without prior conversion of RNA to cDNA is now reported. 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 genomes 1 , 2 , 3 , 4 , 5 , 6 , 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 transcripts 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 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 3′ end heterogeneity and polyadenylated small nucleolar RNAs. This study provides a path to high-throughput and low-cost direct RNA sequencing and achieving the ultimate goal of a comprehensive and bias-free understanding of transcriptomes.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: Biological and medical sciences / Biomedical research / Diverse techniques / DNA microarrays / DNA polymerase / DNA sequencing / DNA, Complementary - genetics / E coli / Fundamental and applied biological sciences. Psychology / Gene Expression Profiling - methods / Genetic engineering / Genetic transcription / Heterogeneity / Humanities and Social Sciences / letter / Methods / Molecular and cellular biology / multidisciplinary / Nucleotide sequencing / Oligoribonucleotides - genetics / Polymerase Chain Reaction / Reaction kinetics / RNA - analysis / RNA - genetics / RNA - isolation & purification / RNA, Fungal - analysis / RNA, Fungal - genetics / RNA, Fungal - isolation & purification / Saccharomyces cerevisiae / Saccharomyces cerevisiae - genetics / Science / Science (multidisciplinary) / Sequence Analysis, RNA - methods / Templates, Genetic