SAN FRANCISCO (GenomeWeb) – Startup 2D Genomics has developed sample prep technology for RNA sequencing that it says will enable researchers to capture and sequence libraries with both small and large RNA molecules.
The Fremont, California-based firm currently has five full-time employees. Pranav Patel and Arek Bibillo, who both previously worked at Pacific Biosciences, co-founded the company in 2016 with an undisclosed amount of seed financing.
Aside from PacBio, Patel also worked stints at 10x Genomics and Theranos, while Bibillo previously worked at Genia, the nanopore sequencing startup that Roche acquired in 2014.
The two had become frustrated by the state of next-generation sequencing library prep, particularly for RNA sequencing. All protocols were too time consuming — taking an entire day in most cases — and lacked the ability to analyze all types of RNA molecules. Kits either target small RNA molecules, like microRNA, or larger molecules, like mRNA, said Patel, 2D Genomics' CEO.
The predominant application of RNA sequencing is to study differential expression, Patel, said. "And, all the different forms of RNA are important, but you have to throw away half because library prep technologies can't do both" small and large RNA.
The other issue, he said, is that most technologies are very inefficient, converting only about 5 percent of the RNA molecules into a sequenceable library.
Current technologies tend to rely on either ligation or random priming, said Bibillo, 2D Genomics' chief technology officer. Bibillo, whose expertise is in enzymology, said the company instead engineered a transposon to give it multifunctionality, allowing it to identify nucleic acid molecules, perform enzymatic activities like replication, and add adapters.
2D has filed a patent on the transposon and its engineering approach, which involves molecular modeling, point mutagenesis, and rational design strategies such as identifying protein structure motifs and domains.
The library prep process is most similar to the template switching method commercialized by Clontech, Patel said, but with some key modifications.
In the template switching method, RNA molecules are reverse transcribed into cDNA and when the enzyme reaches the end of one strand of RNA, it relies on hybridization to a template switching oligo to add the adaptor sequence.
In that method, secondary structures that are present in RNA molecules have presented a challenge for reverse transcription. Typically, heat is used to break apart those secondary structures, Patel said, but higher temperatures prevent the enzyme from efficient template switching to adaptor molecule, so, heat is lowered to enable template switching. The process is not very efficient, Patel said, resulting in fewer RNA molecules being reverse transcribed whereby the enzyme reaches the end of the molecule for efficient template switching.
The 2D Genomics team wanted to improve on these inefficiencies, and as a first step designed a multifunctional transposon that is not heat-dependent. Although the protocol includes one optional heat-dependent fragmentation step, the remaining steps are all performed at ambient temperatures. The transposon is able to reverse transcribe through those secondary structures, Patel said, working "more like strand-displacing enzymes, such as phi29," he said.
In addition, Patel said, it does not rely on a sequence-specific oligonucleotide for template switching. "It can grab another RNA molecule independent of sequence homology," he said.
The company has launched its first kit, 2D Complete RNA, which prepares both small and large RNA in a single prep in less than 3.5 hours, and it is also developing separate products for just small RNA and large RNA. The 2D Complete kit has a list price of $660. Bibillo said the firm has a couple of unnamed early-access industry customers in the liquid biopsy space and is also looking to collaborate with academic researchers.
Patel said the technology should have broad applicability, but that so far the company has focused primarily on the cancer space, including on cell-free RNA for liquid biopsy applications, cancer cell lines, and formalin-fixed paraffin-embedded tissue.
In addition, Bibillo said the firm wants to develop products for in situ RNA sequencing prep as well as single-cell transcriptomics.