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Sequencing Method Detects Previously Unseen RNA Fragments in Plasma

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NEW YORK (GenomeWeb) – A new RNA sequencing method can detect fragments of messenger and long non-coding RNAs in plasma. While still in the proof-of-concept stage, the technology has the potential to turn these extracellular RNA fragments into new biomarkers for use in liquid biopsy.

Phospho-RNA-seq, developed by researchers in the lab of University of Michigan Professor Muneesh Tewari, enzymatically prepares these fragments to be detected by small RNA sequencing protocols. In addition to sample preparation, the technique also relies on new, highly stringent bioinformatics analysis to limit false positive detection.

The researchers published their method earlier this month in The EMBO Journal. They also published data suggesting that phospho-RNA-seq can detect biomarkers associated with biological changes taking place in bone marrow transplant patients.

"This paper is about modifying standard RNA-seq so it becomes sensitive to differing phosphorylation states at the end of RNA fragments," Tewari told GenomeWeb. "What we found is that by paying attention to those states and modifying them, we were able to see thousands of mRNA and lncRNAs that were otherwise invisible." He said the enzymatic modifications were "simple" and should be easy for other researchers to adopt.

Luis Diaz, Jr., head of solid tumor oncology at Memorial Sloan-Kettering Cancer Center and cofounder of liquid biopsy firm Personal Genome Diagnostics, suggested that the new method "uncovers a hidden pool of cell-free RNA" that could be used as biomarkers.

"It's an early paper, but it's fundamentally very strong," said Diaz, who was not involved in the research. "It's the type of tool we can use to uncover biological processes as well as use for some clinical applications." He added, "I personally will jump on this to see if it's reproducible."

Previously, Tewari's lab had worked on developing small RNA sequencing methods. Last year, they described biases in sample preparation kits and in 2008, they published a method to detect microRNAs in blood. Over the years, they had observed that RNA biomarkers in blood, including messenger RNA (mRNA) and long non-coding RNA (lncRNA) had largely been missing.

"For years we've been wondering how to get at those transcripts," Tewari said. "There has been some evidence that they exist, but at the same time, it has been a controversial area. People who have been looking for [mRNA and lncRNA] in the plasma haven't really found them."

Tewari stressed that phospho-RNA-seq is not a replacement for methods to sequence microRNAs. "It's adding another version of small RNA-seq that allows one to see all these other fragments," he said.

The lab's breakthrough came from the hypothesis that RNase enzymes present in the blood were cleaving extracellular RNAs, leaving them with 3' phosphate ends or without 5' phosphate ends. "The typical small RNA-seq methods rely on the fragment that you're cloning to have a 5' phosphate and 3'-OH end," Tewari said. "That's basically required for the method to work."

First, the team designed synthetic RNA with different end modifications. The standard cloning methods didn't work well, Tewari said, and sequencing was unsuccessful. Then, the researchers tried sequencing synthetic oligos after prepping them with T4 polynucleotide kinase, which dephosphorylates 3' ends and phosphorylates 5' ends. "That dramatically allows you to increase recovery of all the fragments," Tewari said. The median number of transcript counts went up at least 100 fold, he said. "They go from unrecoverable to recoverable."

In the paper, the researchers described preparing libraries using the Illumina TruSeq small RNA kit and sequencing on the Illumina NextSeq 500 and HiSeq 2500 instruments.

While most of the mRNA and lncRNA fragments corresponded to annotated RNAs, some did not. "We didn't talk a lot about those in the paper, but quite a few are novel," Tewari said. " Either they're novel because they're not showing up as human or they're novel because they're not well annotated."

The team also had to develop new data analysis pipelines, after they realized they were getting many false positive matches. "We had to be more stringent in our analyses," Tewari. He suggested they may have even detected fewer RNAs than they could have, due to an emphasis on reducing false positives.

Lastly, the researchers applied phospho-RNA-seq to blood samples from patients undergoing bone marrow transplants. These patients often get multiple blood draws per week for periods up to 100 days.

The researchers looked for clusters of transcripts that changed over time and were correlated with each other. Among those, they looked for transcripts that were associated with specific tissue types and whether any tissues were over-represented. Two were: bone marrow and the liver.

"The implication now is that all these RNAs change dynamically in different disease states," he added. His team was not only able to find these RNAs in patients, he said, "but saw that they changed over time in ways that make sense relative to what's happening in the patient."

"We didn't ask whether bone marrow transcripts were changing," Tewari added. "This fell out of analysis. This makes it even more believable for me."Diaz said the method has the potential to impact cancer research, from biomarker discovery to cancer detection and monitoring. "But there needs to be a lot more work done," he said.

Tewari agreed with Diaz's assessment, and said that he'd like to first refine both the laboratory and computational methods used. He suggested that he could increase yield by filtering out reads that don't correspond to mRNA or lncRNA. He also suggested that improving the method would allow researchers to identify more refined cell types.

Phospho-RNA-seq could be applied to cancer research to monitor tissue turnover. "Cancer is a disease where there's lots of cell death, which is presumed to release RNAs into circulation," he said. "Another area of exploration is how exactly do these RNAs get out there?"

The method could also be used in other biofluids containing extracellular RNAs and RNA fragments, including cerebrospinal fluid and urine — which Tewari's lab has plans to study.

Tewari declined to comment on whether he or the university had applied for or obtained intellectual property related to phospho-RNA-seq. "We want to encourage dissemination," he said. "Commercialization is one of those pathways."

Tewari has previously served on scientific advisory boards at CombiMatrix and WaferGen.

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