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Alithea Genomics to Expand High-Throughput Bulk RNA Sequencing Offerings

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BALTIMORE – With proprietary bulk RNA barcoding and sequencing (BRB-seq) technology under its belt, RNA analysis company Alithea Genomics aims to continue growing its high-throughput RNA sequencing market.

The Swiss Federal Institute of Technology Lausanne (EPFL) spinout recently raised CHF 1 million (approximately $1 million) in seed financing and plans to expand its BRB-seq offerings for more sample types and applications later this year.

According to Alithea Cofounder and CEO Riccardo Dainese, the company's core technology BRB-seq was first developed by Chief Technology Officer and Cofounder Daniel Alpern — then his lab mate — to address the budget constraints when conducting high-throughput RNA sequencing experiments.

By rendering the barcoding schemes used in single-cell RNA sequencing to bulk RNA samples, Alpern designed an RNA sequencing method that was both affordable and high-throughput, Dainese said.

Alpern published the method in the journal Genome Biology in April 2019, and soon after, Dainese said, his lab started receiving a lot of requests for BRB-seq as many researchers tried to establish the same protocol in their labs.

By May 2020, the requests had become too many for his lab to accommodate as collaborations, and that's when he and Alpern decided to launch Alithea, officially taking BRB-seq into the commercial realm.

The key difference between BRB-seq and standard RNA sequencing workflows, according to Dainese, is that BRB-seq enables sample pooling into a single tube upstream of the sequencing step.

To achieve that, BRB-seq's workflow starts with barcoding the individual samples. Similar to the barcoding strategy used in single-cell RNA-seq, BRB-seq deploys reverse transcription primers that each contain a unique sample barcode and a ​​unique molecular identifier, which is used downstream to remove PCR duplicates. As a result, an individual barcode is tagged to the cDNA of each sample during the reverse transcription step.

Dainese emphasized that "the key aspect" of the company's IP pertains to the barcodes. Because different barcodes may have different efficiencies, Dainese said the company has to "go the extra mile" to optimize the barcodes both computationally and experimentally, in order to ensure all samples receive consistent sequencing coverage and generate uniform data output. The company has exclusively licensed two patents from EPFL and is also working on two more patents internally, he added.

Once every sample is barcoded, they can be ​​pooled into a single tube for downstream RNA-seq workflows, which is fairly similar to standard RNA-seq sample prep procedures. That said, Dainese noted the main advantage of BRB-seq compared with traditional approaches is that it allows users to execute the rest of the workflow on only one sample. "What this translates into, in practical terms, is a drastic decrease in the amount of reagents and manual operations that need to be performed," he said.

Because of the multiplexing capability of BRB-seq, Dainese said the method is inherently more suitable for high-throughput RNA-seq applications, such as drug screening and RNA analysis within large clinical trials and biobanks.

"The clients that we are now talking to are really those that see the potential for RNA sequencing beyond fundamental research and small-scale studies, and they really want to start using RNA sequencing for big projects," he added.

Alithea currently offers two BRB-seq based products, the Mercurius BRB-seq kit for purified RNA and Mercurius Blood BRB-seq for blood RNA samples.

For the purified RNA kit, the company has barcodes optimized for 24, 96, and 384 samples, catering to customers' throughput desires. However, Dainese acknowledged that this kit is designed to work with high-quality purified RNA, with input amount ranging from 10 ng to 1 μg, and it is "not very well indicated for FFPE samples."

Meanwhile, the blood RNA kit can accommodate up to 96 samples. According to Dainese, the company has integrated a set of globin blockers within the kit chemistry, which promises to "seamlessly" get rid of the globin genes in blood samples.

While Alithea has not disclosed the sticker price for its products publicly, Dainese said that the purified RNA kit cost around $15 per sample and the blood kit around $20 per sample. 

In terms of turnaround time, Dainese said it typically takes two days from RNA to a sequencing-ready library for new users while about a day for experienced users or labs with a certain degree of automation.

Although the BRB-seq kits are designed to only work with Illumina sequencing for now, Dainese said the company is also exploring compatibility with other sequencing modalities such as MGI and nanopore sequencing.

In addition to BRB-seq products, Alithea also provides the technology as a service.

For that, researchers can send extracted RNA samples to the company, where BRB-seq will be run. As part of the standard service, the company will also return a basic sequencing report, containing sequencing and alignment statistics as well as a gene count matrix.

Similarly, both the company's kit or service customers will have access to Alithea's cloud-based data-analysis pipeline, which allows them to produce a report displaying the main alignment statistics and a gene count matrix that can be used for offline gene expression analysis.

Dainese said the company has garnered sizeable interest for the service, but that Alithea intends to place more emphasis on marketing its products.

"What we want to do moving forward is to really promote the usage of BRB-sequencing and the kits by other service providers," he said.

Still, Dainese does not think the company will eliminate the service model altogether. Instead, it plans to use it as part of the learning process for new technologies.

To that end, with the seed funding, Dainese said the company will continue expanding its BRB-seq product offerings with the aim of releasing two new products in the second half of this year.

One of the new products will be the BRB-seq cell lysate kit, which promises to skip the RNA extraction step and achieve BRB-seq on cell lysates directly, according to Dainese. The other product will be a high sensitivity BRB-Seq kit, which is engineered to work with low RNA input samples — such as FFPE, small organoids, and single cells — that the company's current products cannot really tackle.

Before launching these new products, Dainese said the company will also host a beta testing program, where the first batch of kits is delivered to early-access users for feedback. So far, he said, three global pharmaceutical companies have expressed interest in trying out the cell lysate kit for drug screening, but he did not disclose which companies.

The company also plans to scale up operations as well as sales and marketing efforts. According to Dainese, the company currently has 10 full-time employees and is "probably going to expand with three or four more people this year."

Dainese considers Austria-based biotech company Lexogen as Alithea's "closest competitor" in terms of technology. Besides that, he said the company also faces competition from traditional big players in RNA-seq sample prep, such as New England Biolabs, Thermo Fisher Scientific, and Qiagen.

To stay competitive, Dainese said the company aspires to eventually achieve full-length RNA sequencing with BRB-seq, which currently remains a 3'-end RNA sequencing technology. While 3'-end sequencing is great for counting genes, it is not suited for profiling splicing variants or isoform detection, he pointed out.

To that initiative, Dainese said the next iteration of the company's R&D efforts will be toward solving the "significant technical challenges" for full-length RNA sequencing while making it compatible with BRB-seq's multiplexing ability.

Additionally, as BRB-seq becomes increasingly mainstream for high-throughput applications, Dainese said the company also recognizes that it is "especially important" for the company to provide a "turn-key solution" for BRB-seq. That said, the firm is also collaborating with liquid handling providers to automate the BRB-seq workflows.

Lastly, despite the rise of single-cell RNA sequencing technologies, Dainese thinks there is still a great demand for bulk RNA sequencing, as many bottlenecks still persist in the single-cell field, especially when it comes to sample throughput, data handling, and the application value.

"Our goal is going to be to identify these bottlenecks, create a niche, and that's where we want to excel and dominate," he said.

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