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Life Tech Developing DNA/RNA Combo AmpliSeq Workflow


NEW YORK (GenomeWeb) — Thermo Fisher's Life Technologies is developing a sample prep workflow to isolate, prepare, and sequence both DNA and RNA from the same sample.

Researchers from the company presented a poster describing the technique at the American Association for Cancer Research earlier this month. In a follow-up interview with In Sequence, staff scientist Angie Cheng said the researchers decided to design the protocol because the firm had been getting lots of requests for kits that could purify DNA and RNA from the same sample, especially from researchers who "were working with precious samples like tumor biopsies or [formalin-fixed paraffin-embedded] blocks, and wanted to maximize the information they could recover."

At the same time, Life Tech had been working on developing AmpliSeq cancer panels both for analyzing point mutations in DNA and specific gene fusions from RNA, Cheng said. So, they decided to develop technology that would do sample prep for DNA and RNA sequencing libraries from the same sample, and combine the gene fusion and point mutation AmpliSeq panels.

The ability to isolate and analyze both DNA and RNA from the same sample has several advantages, Cheng said. For one, it reduces the amount of sample material needed, which is particularly important when working with cancer samples, which tend to be FFPE samples. Additionally, she said, if different biopsy samples are used for both DNA and RNA analysis, there is always the risk that the two samples may have a slightly different genetic makeup due to the heterogeneous nature of tumors. But, analyzing DNA and RNA from a single sample helps assure that the DNA and RNA correspond with each other.

Cheng said that to design the workflow, the researchers had to address three different areas: getting DNA and RNA from the same sample, developing the AmpliSeq panel that could target both DNA and RNA, and then the sequencing portion.

For sample prep, Cheng said the researchers used the existing silica-based column technology and that the main hurdle was to avoid cross-contamination of DNA and RNA. In order to make it robust for FFPE samples, she said the researchers had to "fine tune" the Ambion RNA isolation protocol.

The AmpliSeq technology has different protocols for dealing with RNA and DNA, Cheng said. For example, when preparing RNA for sequencing, a reverse transcription step must be performed first. Additionally, the clean-up steps for DNA and RNA prep were different, she said. "Now we've worked to unify that," Cheng said.

For the combination workflow, the reverse transcription step is performed up front, she said, and then researchers can follow the same AmpliSeq protocol regardless of whether it is DNA or RNA. The cleanup protocols and quantitation steps are both the same now.

The AmpliSeq DNA and RNA libraries are barcoded and can be run on the Ion Torrent PGM. Cheng said that the researchers worked with the 318 chip and were able to multiplex two DNA and two RNA libraries and sequence to an average of 500x coverage. In the poster, they demonstrated the combo panel on a non-small cell lung adenocarcinoma, testing the technique on FFPE samples, as well as fresh tissue and cells.

They combined the AmpliSeq Colon and Lung hotspot panel consisting of 90 amplicons that cover 22 genes related to lung and colon cancers and the AmpliSeq RNA Lung Fusion panel covering 40 fusions corresponding with the ALK, RET, and ROS1 genes. Life Tech developed the panels in collaboration with the OncoNetwork Consortium, a Life Tech-coordinated effort to develop AmpliSeq cancer panels that consists of groups from Radboud University Medical Center in the Netherlands; Cancer Research Center of Mercogliano in Italy; the genetics and molecular biology division of pharmaceutical company Viollier in Switzerland; the University of Warwick in the UK; the Institut Gustave Roussy in France; the University Paris Descartes Center in France; and St James' Hospital in Dublin.

Cheng said the main thing the team had to figure out when sequencing the combined DNA and RNA libraries was how many reads would cover each panel, and whether the best results would be obtained by devoting equal read counts to each library.

Cheng said the Life Tech team thought that researchers may want more reads devoted to the DNA library since the library contains more amplicons than the RNA library and since researchers tend to want very high coverage to look for low-frequency point mutations.

The Life Tech team figured out that including equimolar pools of the DNA and RNA in one run on the 318 chip yielded slightly more reads for the DNA library — about 55 percent of the reads aligned to DNA.

Cheng said the Life Tech team is still "ironing out the unified AmpliSeq protocol," including determining the optimal ratio of DNA to RNA, after which the firm will launch as a commercial product.

Moving forward, she said that customers would be able to purchase the DNA/RNA isolation kit and use it with both fixed or custom designed AmpliSeq panels.

Others have also realized the benefits of being able to sequence DNA and RNA in one assay. Earlier this month, Paradigm launched a cancer diagnostic that uses next-gen sequencing on the PGM to analyze both DNA and mRNA. The non-profit entity did not provide many details about its custom-designed targeted panel, but said that it does not use the AmpliSeq technology.

In addition, the National Cancer Institute is developing a DNA/RNA AmpliSeq panel that runs on the PGM for its NCI Molecular Analysis for Therapy Choice trial.

Cheng said that although the company has not yet set a price for the product, she anticipates that it will save customers both time and money. "It's a cost savings because you can multiplex," she said. "From one sample, you can harness both DNA and RNA." Additionally, the "AmpliSeq protocol is unified [for both analytes], so requires no extra reagents, and then you can sequence on the same chip."