NEW YORK (GenomeWeb News) – Researchers from Helicos BioSciences today published a proof-of-principle study illustrating the feasibility of doing direct RNA sequencing using its single molecule sequencing technology.
The team used a prototype Helicos instrument to directly sequence RNA from Baker's yeast, Saccharomyces cerevisiae without first converting it to complementary DNA. In the process, they also uncovered heterogeneity at the 3' ends of many S. cerevisiae transcripts, as well as evidence that at least some small nucleolar and ribosomal RNAs are polyadenylated in yeast.
The findings appeared in a letter in the advance, online edition of Nature today. Senior author Patrice Milos, vice president and chief scientific officer at Helicos, told GenomeWeb Daily News that researchers at Helicos are currently tweaking the direct RNA sequencing protocol for use on the company's commercially available sequencer, the Heliscope.
As researchers have increasingly appreciated the importance of RNA, Milos explained, a variety of approaches have sprung up to analyze it. But despite the advent of sequencing-based approaches for examining RNA, she and her co-authors argue, many methods still rely on reverse transcription to create cDNA — a step that they say introduces errors and inefficiency.
"[T]here is an ever-growing need for a method that would not be subject to the difficulties associated with [reverse transcriptase] behavior, amplification, ligation, and other cDNA synthesis/sample manipulation steps," the researchers wrote. "A method allowing a comprehensive and bias-free view of transcriptomes using minute quantities of total RNA obtained from as few as one cell with no pre-treatment would stimulate great advances in the delineation of complex biological processes and be applicable across all biomedical research areas."
In an effort to adapt their sequencing-by-synthesis reaction for directly sequencing RNA, Milos and her co-workers optimized everything from the polymerase enzyme used to the buffer conditions and proprietary fluorescent nucleotide analogues (dubbed "virtual terminator," or VT, nucleotides) used. In general, the method relies on capturing polyadenylated RNAs on a poly(dT)-coated surface and doing the sequencing-by-synthesis step using the Escherichia coli poly(A) polymerase I enzyme.
The team first tested the approach by sequencing a 40-base RNA oligonucleotide. In that experiment, nearly half of the aligned reads — 48.5 percent — were 20 nucleotides or longer. The longest error-free read was 38 nucleotides.
They then turned their attention to the S. cerevisiae, a model organism that has been fairly well characterized genetically. Because much of S. cerevisiae's RNA is naturally polyadenylated, the researchers noted, they did not add poly-A tails to the yeast RNA before sequencing it.
In their yeast RNA sequencing experiment, the team generated 41,261 reads that were 20 or more nucleotides long. Almost half of these — 48.4 percent — mapped to the yeast genome. And more than 90 percent of the aligned reads were located within 400 nucleotides of known yeast open reading frame 3' ends.
In the process, the researchers detected unexpected heterogeneity at the 3' ends of yeast RNAs, Milos noted. They also found evidence suggesting that at least some yeast ribosomal RNAs and small nucleolar RNAs, or snoRNAs, are polyadenylated.
The researchers reported that their direct RNA sequencing method currently has an error rate of about four percent — mostly due to deletions caused by dark bases. The insertion error rate was one to two percent, while substitutions occurred with a rate of just 0.1 to 0.3 percent.
The team is doing similar studies and developing the methodology for directly sequencing RNA in mammalian cells as well, Milos said.
And although the study was done using a prototype Helicos instrument, she added, the company is currently working to develop the direct RNA sequencing protocol for its commercially available instrument. "Our focus right now is really optimizing the methodologies for the Heliscope," Milos said. That protocol is expected to be available sometime next year.
Last week, GWDN's sister publication In Sequence reported that Pacific Biosciences is using its own instrument for both direct RNA sequencing and methylation sequencing — work presented by Steve Turner, PacBio's chief technology officer, at the recent Cold Spring Harbor Laboratory Personal Genomes meeting. Turner said PacBio plans to make direct RNA sequencing available for its platform within a year of platform launch.