NEW YORK –
Loop Genomics is gunning to take over one of the major remaining applications of Sanger sequencing: bacterial clonal isolate analysis.
The firm's LoopSeq Solo sample preparation technology for next-generation sequencing, offered commercially as both a kit and a service since early August, is a way to get a single, definitive readout from a particular sample, often a bacterial colony, that's longer than what can fit on a single Illumina read.
To do this, the company first attaches PCR barcodes to samples in each well of a plate and pools the samples. It then uses proprietary enzymes to randomly copy each barcode within the molecule it is attached to. After sequencing, short reads that share the same barcode are combined into a single synthetic long read.
Getting the single readout for a sample — a plasmid, a gene fragment, a PCR product — is a process that can be done with standard short-read NGS, but with an expensive and time-consuming catch. "If you have 10,000 samples, you would run the [sample prep] 10,000 times, one for each sample," said Tuval Ben-Yehezkel, Loop's CEO. "That's prohibitively expensive and it's preventing people from scaling their projects."
Loop's new kit enables up to 6,000 samples to be prepared simultaneously, all in the time it would take to prep one sample with a different kit. "For customers sequencing thousands of samples on a routine basis, the cost savings is larger than an order of magnitude," Ben-Yehezkel said.
As part of an early access program, Loop provided LoopSeq Solo as a service to a customer looking to get more than just one read out of each sample. General Automation Lab Technologies (GALT), based in San Carlos, California, is developing the Prospector, an automated, benchtop microbiology platform that generates human microbiome isolates at a scale previously unseen with traditional methods. Loop's tech solves a major problem with Sanger sequencing, which is the failure of the assay, often due to mixed cultures; Loop estimated that up to 20 percent of clonal samples would fail using the Sanger method.
"On the rare occasion we're not getting pure isolate, where we're coculturing species together, sequencing would immediately fail" using the Sanger method, said Crystal Emery, an application scientist at GALT. With Loop, that's no longer a problem and she can see which samples are mixed.
"It's allowing us a faster and more reliable access to the isolates that we're providing to our customers," she said. "Also, their sequencing quality is excellent. For 16S ribosomal RNA sequencing — used to identify species and strains of bacteria — Loop's tech provides a consistent, full-length gene. You get a high confidence in the species you're declaring," she said.
The new library prep method is built on previous Loop synthetic long-read technology. Since 2018, Loop has secured distribution deals bringing that tech to Australia, China, Denmark, France, Germany, the Netherlands, Japan, Taiwan, Japan, Sweden and the UK.
"The only difference is that this time we're not constructing a single long read per molecule but a single long read per sample," Ben-Yehezkel said. The firm spent about a year developing LoopSeq Solo, he noted.
Loop did not disclose which enzyme the method uses to distribute the barcodes, but said that it wasn't a transposase. "It's similar in the sense that we're copying and pasting a sequence from one molecule to another molecule," Ben-Yehezkel said.
The San Jose, California-based firm raised $8 million in a 2018 Series A financing round and is not actively fundraising now as it is comfortable with its cash runway, he said. He noted that the firm is growing its headcount to support its products and services, with an estimated 50 percent growth year over year, both this year and going forward.
The pandemic hasn't severely affected the business operations, Ben-Yehezkel said, and the firm is back to work at full capacity. Loop has even launched a SARS-CoV-2 sequencing service.
Loop declined to specify a price for LoopSeq Solo, saying that per-sample cost was highly dependent on how many samples were being sequenced and the length of the desired readout. For researchers doing just a few clones, Sanger may still be more cost effective, Ben-Yehezkel said. "But starting with a plate of 96 samples, or more, it just wouldn't be," compared to LoopSeq Solo, he added.
Emery said the cost was "comparable to Sanger," but using Loop's service "saves so much time […] and you get the advantage of additional data that you wouldn't normally [get with Sanger.]"
"We have been giving them plates of liquid cultures and they give back a full data package where they do some raw data and also give you all the information in terms of quality scores and taxonomic assignments," as well as data visualizations, she said. "You can choose the range of how in depth you want to get. If you trust its face value, it's easy to interpret. If you want to get more in depth, you can investigate it on your own, too."
Loop has also been active in making sure GALT's scientists understand the data coming out of the service. "It's very clear, compared to some other technologies," Emery said. "It feels like data you can interpret relatively easily." Moreover, "they've been really flexible to work with," she said. "They're very dedicated scientists and always interested in what's going on in the field."
Using Loop's service has "been very helpful in moving our projects much faster," Surekha Karudapuram, GALT's senior director of marketing, added. "We're just generating so much in our labs, that opportunity to have someone else take care of the process has been really nice."
So far, most customers have been applying the kit or service to bioengineering projects such as protein design, gene editing, or antibody development, Ben-Yehezkel said. But Loop Chief Commercial Officer Tony Lialin suggested future uses could include prenatal testing or other diagnostics where the goal is to look at the same genes over and over again. "We are working with a group looking at SMA1 and 2 [genes analyzed in carrier screening] using our low-volume Amplicon kit, with eyes on the new kit for higher volume," he said.
Ben-Yehezkel suggested that the technology is changing not only the scale of customer projects but also design paradigms. "Within the field of protein and antibody design, the limitations of Illumina and Sanger sequencing are so built into how people think that their designs are, at the outset limited to what you can read through [with] NGS," he said, usually no more than 500 bp within a gene that could be up to 3,000 bp long. With Loop's tech, "you can design any part, not just the part that's long enough to read," he said.