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Japanese Startup BitBiome Builds Single-Cell Bacterial Sequencing Services Business


NEW YORK – Fresh off a ¥700 million ($6.6 million) Series B financing round and several peer-reviewed publications, Tokyo-based startup BitBiome is looking to grow the market for its single-cell bacterial sequencing services.

Spun out of Waseda University in 2018 and funded largely by University of Tokyo Edge Capital Partners, the firm has exclusive rights to a sample preparation technique that uses gel capsules to prepare bacteria for whole-genome analysis using off-the-shelf microfluidics. The key to the firm's technology lies in amplifying DNA, BitBiome CEO Sunao Fujioka said. At low temperatures, the gel solidifies around single cells before lysis, allowing amplification to happen within each capsule. Gel capsules are then run through a fluorescence-activated cell sorter to separate out empty capsules prior to whole-genome or amplicon sequencing.

The technology offers a "deep dive" into the genomes of bacteria in a complex sample — often from skin, stool, or environmental microbiomes — especially those of unculturable species, offering strain-level resolution that metagenomics usually can't deliver, Fujioka said. The firm's Bit-Map platform delivers an average of 22 genomes per sample for stool samples, compared to six for shotgun metagenomics, and the data result in a "higher number of quality genomes," he said. "We can focus on who's really doing what and who's responsible for a disease biomarker or a druggable target."

As proof of concept, BitBiome researchers ran a soil sample from a strawberry farm, obtaining draft genomes from two potentially novel, uncultivated bacterial species in the class Gammaproteobacteria, which includes important human pathogens Salmonella, Yersinia pestis, and Escherichia coli. Last month, they published their results in Microbiology Resource Announcements.

So far, the firm's business has centered around collaborations with academic research institutions and biotechnology firms in its home country. In 2019, BitBiome signed a collaborative research agreement with the National Cancer Center Japan to analyze human gut bacteria in a study of cancer patients. Fujioka declined to disclose its private-sector partners, citing confidentiality agreements. But expanding into other markets, especially the US, is now a priority.

"They seem to have some promising results," said Ramunas Stepanauskas, director of the single-cell genomics center at the Bigelow Laboratory for Ocean Sciences. In addition to its own work in the field, Stepanauskas's lab also performs services for other researchers, from isolation to bioinformatics. Recovering whole genomes from bacterial cells has been very difficult, he said, but BitBiome's method is one of several that are improving the fraction recovered. "There still needs to be larger-scale, independent validation studies," he said, adding that while BitBiome improves the initial capture of cells and amplification of their genomes, it uses a lower-throughput method, usually FACS, to move the DNA to a plate for the downstream steps.

"So far I haven't seen a breakthrough in the field with these microfluidic techniques that show an obvious advantage in what you can get out of a cell, but in the long run, there will be an advantage in cutting costs or increasing throughput in the same way microdroplets have helped with single-cell transcriptomics," he said.

BitBiome plans to commercialize sequencing of single-cell amplified genomes in gel beads (SAG-gel), developed at Waseda University by Masahito Hosokawa, who is also BitBiome's CSO. Originally developed for analyzing gut microbiomes, the method was described in a series of papers published in Scientific Reports in 2018.

In addition to its recent financing, the firm raised approximately ¥350 million in a 2019 Series A funding round.

BitBiome also offers 16S ribosomal RNA sequencing and shotgun metagenomics to complement its single-cell platform. "We are not the company to replace existing metagenomics, but to add value to those approaches," Fujioka said.

While single-cell transcriptomics has taken off in recent years, single-cell bacterial genomics, which has been around just as long, hasn't received the same attention.

From a research perspective, single-cell bacterial genomics offers the best chance at capturing the full picture of microbial life on Earth. "If we can’t sequence individual cells, we don’t really have a clue of what [their] gene content is," Stepanauskas said. "You don’t catch that in just a single metagenome assembly." In addition to providing data that can be applied towards fundamental biological questions, genes found through single-cell sequencing could be harnessed for industrial or biomedical applications.

The current state of the art for single-cell microbial genomics is a collection of different technologies, including isolation, sample prep, sequencing, and informatics, Stepanauskas explained. "Each of these steps have their own trajectories and development," he said.

BitBiome's advances appear to be focused on isolation and sample prep, although Fujioka noted that the firm is also focused on the data analysis side. The firm can assemble a genome de novo for every cell; however, "if there's a reference genome, we can quickly use that to create the whole genome," he said. In fact, the firm is seeking a patent for a method to merge analysis of single-cell DNA data with a reference database. BitBiome is building an internal database to efficiently collect contigs and assemble them into draft genomes. Customers receive a report on the different genomes found in their sample and their gene content.

Whole-genome data costs less than $100 per cell, and running a sample can cost customers $10,000. "It really depends on how many cells they want," Fujioka said. He noted the firm tries to optimize the study design, in terms of number of samples and cells, within the budget constraints of the customer.

Some applications Bit-Map has been used for include functional pathway analysis, drug resistance gene identification, and environmental analysis.

"The addressable market is huge," Fujioka said. "There are tons of microbes still unknown." And without a cell-by-cell interrogation, "they're just a bunch of unknown species with unknown functions."