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10x Genomics Previews New Single-Cell Assays at AGBT


ORLANDO (Florida) – 10x Genomics this week previewed three new single-cell assays that it plans to launch this year for its Chromium Controller platform: single-cell CNV, single-cell ATAC-seq, and single-cell feature barcoding.

The company showcased the assays at the Advances in Genome Biology and Technology meeting held here. All three assays are already in the hands of early-access customers, and pricing information will be revealed closer to their launch.

The single-cell CNV assay, which will become available by mid-year, lets users analyze copy number variants in a few hundred to about 5,000 single cells at a time, and several samples can run in parallel on each microfluidic chip. This assay will help researchers uncover the genomic heterogeneity of tissues or cell lines, and to understand clonal evolution, which are all important in cancer.

The assay involves a new, two-step approach, called CBGB for "cell beads, gel beads," and requires two chips. In the first step, cells are trapped individually in a gel matrix, so-called cell beads, where they are lysed and their proteins are digested and washed out, while their DNA remains inside. After that, each cell bead is encapsulated together with a gel bead that contains a library of barcoded oligos, followed by the usual 10x process. The CBGB approach will "enable a whole host of multi-step reactions on our platform that were previously inaccessible," said Michael Schnall-Levin, 10x Genomics' VP of product, R&D, and strategy, during a company-sponsored workshop.

For proof of concept, 10x Genomics researchers spiked either 5 percent or 1 percent cancer cells into a background of diploid cells and were able to identify the malignant cells because of their copy number variations. In addition, they applied the assay to a well-known cancer cell line, COLO-829, and found that it is not a single cell population but is actually mosaic for copy number variants.

The company has also applied the assay to single cells isolated from breast cancer tissue and found a lot of copy number heterogeneity, as is typical for many cancer samples.

An early-access customer for the assay, Hanlee Ji's group at Stanford University, also showed a poster at the meeting describing how they measured copy number variants in 710 cells from a primary gastric cancer tumor and found three subclones with distinct CNV patterns.

The single-cell ATAC-seq (assay for transposase-accessible chromatin using sequencing) assay, which will be available early in the fourth quarter, allows customers to study chromatin accessibility in up to about 10,000 single cells at a time. Many of the company's existing single-cell RNA-seq customers asked for this assay, which will provide complementary data to single-cell gene expression analysis, Schnall-Levin said. Also, large projects like the Human Cell Atlas are looking to run single-cell ATAC-seq assays "if they can find a good solution," he added.

During the workshop, he showed data for a cell line demonstrating that the single-cell ATAC-seq assay recapitulates the results of an ATAC-seq bulk experiment. In another study, the company generated data on 1,000 single cells derived from blood and was able to recover all major cell types present in the sample. For each cell, they built profiles of open and closed chromatin, which can help researchers understand gene regulation.

Finally, the single-cell feature barcoding assay, also scheduled to be available early in the fourth quarter, will allow users to profile a specific cellular feature, such as cell-surface proteins or CRISPR guide RNA perturbations, at the same time as gene expression. The features are detected using oligo barcodes, which can be conjugated to antibodies, for example, or added to guide RNAs. This assay will also be able to analyze up to about 10,000 cells in parallel.

The idea for this assay is not new — customers have already used their 10x platform for simultaneous dual measurements in single cells, Schnall-Levin said, but the company now plans to offer the assay in a more standardized format to enable other labs.

Also, other companies are pursuing similar approaches. BD Biosciences, for example, showed in a poster at the conference that it can use targeted RNA-seq and oligo-conjugated antibodies to profile both RNA and proteins in single cells, an application for its Rhapsody platform that it talked about last year.

Regarding the 10x Genomics assay, researchers at the New York Genome Center last summer published a method called CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing), for which they used the 10x platform as well as an in-house-developed microfluidic device to measure both the transcriptome and 10 cell surface proteins in thousands of single cells.

Also last summer, Merck researchers published a method called REAP-seq (RNA expression and protein sequencing assay) that let them measure gene expression and quantify cell-surface proteins with 82 barcoded antibodies in single cells at the same time.

For the simultaneous measurement of RNA and proteins, 10x Genomics will provide protocols for conjugating barcoded oligos to antibodies and will work with partners who can provide pre-conjugated antibodies. But customers are also free to conjugate other molecule they wish to profile to the oligos.

For example, several research teams in late 2016 published related methods that combine CRISPR-based single-cell perturbation screens with single-cell RNA sequencing. This approach, called Perturb-seq, tags each guide RNA with an oligo.

According to Schnall-Levin, the single cell feature barcoding assay will "enable a whole host of applications" in the future that will go hand in hand with the company's existing single-cell transcriptome and single-cell immune profiling assays, and 10x will talk more about these applications over the coming year. "We don't think we even know all the things we can do with it," he said.

One of the applications could be to map T-cell and B-cell receptor sequences to their antigens in a massively parallel format. For proof of concept, 10x Genomics researchers have spiked T cells with known specificity to the Epstein-Barr virus into a sample of peripheral T cells. They also attached a feature barcode to an MHC-viral peptide tetramer and analyzed the cells with the company's recently launched single cell immune profiling assay. They recovered almost 500 T cell clonotypes of matched TCR alpha and beta chain sequences and identified the T cell clone that was specific to the viral peptide.

There might be practical limitations to how many antigens can be assayed like this in parallel, Schnall-Levin said, but the company does not know yet what they are and counts on its customers to explore the possibilities the assay offers. "We have a lot of customers who want to do this," he said. "You can imagine grabbing every neoantigen from a cancer patient, synthesizing those and presenting those, or people screening whole libraries of every pathogen peptide known. The scale of it could be immense."