This article has been updated with additional information from Bio-Rad's lawsuit against 10X Genomics.
NEW YORK (GenomeWeb) – 10X Genomics launched its GemCode platform at the Advances in Genome Biology and Technology meeting in Marco Island, Fla., last week and provided more detailed information about the technology behind it.
As the company, which recently raised $55.5 million in a Series B financing round, gears up to commercialize the platform, it is fighting separate lawsuits from two firms — RainDance Technologies and Bio-Rad Laboratories — claiming that 10X violates their intellectual property rights.
The GemCode platform, which allows users to obtain long-range genomic information from placing short sequence reads in the context of large DNA fragments, has been used by several early-access customers, some of whom presented at AGBT. Its applications include haplotype phasing, structural variation analysis, and de novo genome assembly.
The GemCode instrument, about the size of a benchtop PCR machine, partitions long DNA fragments, combines them with barcode-containing oligonucleotides on hydrogel beads, and places them into droplets, using chip-based microfluidic technology. The company is accepting orders for the $75,000 instrument now and anticipates shipping the first units in the second quarter. Reagent cost per sample will be $500, and the firm will ship kits with gel beads and library reagents for 16 samples, and kits with chips and associated reagents for 48 samples.
Each chip can process up to eight DNA samples at a time, performing partitioning and reagent loading in about five minutes. This is followed by an amplification step that creates short barcoded DNA molecules, which are then pooled to generate standard Illumina sequencing libraries. After sequencing, reads with the same barcode can be used to computationally reconstruct longer DNA fragments.
What sets the 10X Genomics technology apart from other approaches to reconstruct long-range information from short reads, such as Illumina's TruSeq Synthetic Long-Read technology, originally developed by Moleculo, is its scale: the technology can partition DNA into more than 100,000 fractions, each containing about 0.3 percent of the genome, and has 750,000 different barcodes available.
Another distinguishing feature is the low amount of DNA required as input – 1 nanogram is sufficient, with an average DNA fragment length of 50 kilobases – which makes it compatible with projects that only have limited material available. Despite the low DNA input requirements, the complexity of the resulting sequencing libraries remains high, according to the company.
At the heart of the technology are 54-micrometer hydrogel beads, each studded with millions of oligos carrying a 14-base barcode from a 750,000-barcode library as well as sequences required for standard Illumina library construction.
On the chip, a "gel bead flow cytometer" connected to a droplet generator first combines individual gel beads with DNA template molecules and enzymes. At a second junction on the chip, beads and reagents are encapsulated into droplets in oil, so-called GEMs or "gel-beads in emulsion." Ninety percent of the time, a droplet contains a single gel bead, and each droplet obtains less than 10 femtograms of template DNA.
Inside the droplet, the gel then dissolves, releasing millions of barcoded oligos into solution, where they hybridize with the template DNA.
Off chip, the company then performs a "highly efficient biochemistry" to generate molecularly barcoded DNA fragments, according to Ben Hindson, president, co-founder, and CSO of 10X Genomics, who gave a presentation at AGBT last week. The process, a cycling reaction, "prevents typical artifacts you see with other amplification processes," he said.
After breaking the emulsion, all barcoded fragments generated from a sample are pooled and processed to generate a standard Illumina sequencing library. Alternatively, it is possible to perform exome capture on the barcoded fragments first, prior to library construction.
For the data analysis, 10X Genomics provides software that combines standard alignment and variant calling tools with barcode processing, phasing, structural variant calling, and visualization tools, such as a haplotype-resolved genome browser. The environment is open source and the company encourages the community to contribute to the software suite, Hindson said.
Resulting from the analysis are so-called "linked reads," with dots representing barcoded reads that are connected to each other, like beads on a string.
One application of the technology is haplotype phasing of whole human genomes and exomes, which 10X Genomics has performed on a number of standard reference samples, including HapMap sample NA12878. For the genomes, the size of N50 phase blocks is on the order of megabases, Hindson said, and the percentage of phased SNPs and fully phased genes is high. Results for exomes are similar and represent "probably the first example ever of a phased exome," he said, noting that no statistical phasing was used in the analysis.
The technology can also be applied to phase the complex HLA region of the human genome, and results the company obtained for NA12878 agreed completely with known phasing information for several HLA genes in that sample.
10X researchers were also able to call a number of structural variations in NA12878, including five large-scale deletions, and found that three large-scale deletions previously detected with short-read sequence data are probably false positives.
In cancer genomes, the technology has promise for calling gene fusions. For example, 10X Genomics has been able to call an EML4/ALK fusion in a lung cancer cell line and found that part of the ALK gene had integrated into a different chromosome.
Early-access customer reports
Several early-access customers presented early results from the GemCode platform at the meeting.
Hanlee Ji of Stanford University School of Medicine, for example, reported on sequencing and phasing the genomes of primary colorectal adenocarcinoma samples. According to Ji's conference abstract, he was able to product haplotype blocks greater than 11 megabases in size, with an N50 of 1.2 megabases, and found "somatic genetic aberrations that would otherwise be missed by traditional short reads, some with clinical significance."
David Jaffe of the Broad Institute reported at the meeting how the 10X Genomics technology has helped his group with the de novo assembly of cancer genomes using the DISCOVAR algorithm by resolving assembly graphs. A single linked read, for example, more than 100 kilobases in size, enabled them to trace a path through an assembly graph, he said, allowing them to find somatic mutations that were "not detectable in other ways."
Likewise, Levi Garraway from the Dana Farber Cancer Institute mentioned in his presentation at AGBT that the 10X Genomics technology has helped him resolve complex rearrangements in cancer genomes.
Besides cancer, Hindson mentioned that the company recently started collaborating with the Center for Mendelian Genomics at the University of Washington, preparing "high complexity samples" for Jay Shendure's group from blood, buccal, and archived buccal samples where little material was available.
While many AGBT attendees told GenomeWeb they are interested in testing the 10X Genomics technology, two companies claim it violates their IP rights.
As reported earlier, RainDance filed suit in the US District Court for the District of Delaware last month, accusing 10X of infringing six of its patents, exclusively licensed to RainDance by the University of Chicago.
Bio-Rad already filed suit against 10X last fall. In a complaint filed Sept. 22 with the Superior Court of the State of California for the County of Contra Costa, Bio-Rad claimed ownership of intellectual property described in patent applications from 10X, and said that 10X officials misappropriated trade secrets from Bio-Rad.
10X officials, Bio-Rad said, violated the terms of their employment and non-competition agreements, which were negotiated as part of Bio-Rad's acquisition of QuantaLife. The three 10X co-founders — CEO Serge Saxonov, CSO and President Ben Hindson, and CTO Kevin Ness — are all former QuantaLife employees and initially stayed with Bio-Rad after the $162 million acquisition, which was announced in October of 2011.
According to the complaint, Saxonov and Hindson left Bio-Rad in April of 2012 and founded Avante Biosystems in July, while Ness joined the firm in August. The company later changed its name to 10X Technologies, and is now called 10X Genomics.
Shortly after its foundation, 10X started to file US patent applications, which Bio-Rad claims disclose ideas and know-how relating to partition technology that were originally developed at QuantaLife and Bio-Rad. Bio-Rad only became aware of these applications when they were published, starting in February 2014.
Some of the patent applications, Bio-Rad maintained, improperly disclose concepts and inventions that were conceived at Bio-Rad, and the applications demonstrate that 10X plans to develop products that will compete with Bio-Rad's.
Several other Bio-Rad employees who knew about the firm's confidential plans for expanding applications for the partitioning technology, reagent formulations inside droplets, and emulsion chemistries, joined 10X between 2012 and 2014, according to the complaint.
Bio-Rad asked the court to declare that it owns the patent applications filed by 10X and to impose an injunction against 10X that would require the company to return the trade secrets to Bio-Rad and prohibit 10X from disclosing them.
In addition, Bio-Rad asked for punitive damages and attorneys' fees.
"We firmly believe there is no merit to the company's claims," Saxonov told GenomeWeb.