Direct RNA sequencing.
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, 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. 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.
Ozsolak
Fatih
F
Helicos BioSciences Corporation, One Kendall Square, Cambridge, Massachusetts 02139, USA. fatihozsolak@gmail.com
Platt
Adam R
AR
Jones
Dan R
DR
Reifenberger
Jeffrey G
JG
Sass
Lauryn E
LE
McInerney
Peter
P
Thompson
John F
JF
Bowers
Jayson
J
Jarosz
Mirna
M
Milos
Patrice M
PM
D018076:DNA, Complementary / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods; D009843:Oligoribonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction; D012313:RNA / Q000032:analysis* / Q000235:genetics* / Q000302:isolation & purification; D012331:RNA, Fungal / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017423:Sequence Analysis, RNA / Q000379:methods*; D013698:Templates, Genetic
D018076:DNA, Complementary; D009843:Oligoribonucleotides; D012331:RNA, Fungal; D012313:RNA