News: AACR Recap

The 108th annual American Association for Cancer Research (AACR) meeting was held April 1–5 in Washington, DC. The conference had record attendance of 21,900 registrants. The total number of exhibitors was 497.

The exhibit was a showcase for several new products, in particular, products for NGS. 10x Genomics launched the Chromium Single Cell V(D)J Solution for profiling full-length paired V(D)J transcripts from hundreds to millions of lymphocytes. (From nature.com, “VDJ recombination is the process by which T cells and B cells randomly assemble different gene segments—known as variable (V), diversity (D) and joining (J) genes—in order to generate unique receptors [known as antigen receptors] that can collectively recognize many different types of molecule.”) Antigen specificity is thus determined by two co-expressed genes, the heavy and light chains of the B-cell receptor, and the alpha and beta chains of the T-cell receptor.

This is the 10x’s second reagent system for single-cell analysis. The company chose this application because the ability to more completely identify and characterize T and B cells by single-cell sequencing will reveal the true clonality and diversity of the adaptive immune system; thus, lending insight into a wide range of applications, including the development of immune-oncology drugs for targeted cancer therapy. The reagent system for T-cell analysis is available now; the reagents for B-cell analysis should be released later this year. Lymphocytes, Peripheral Blood Mononuclear Cells (PBMCs), cell lines, FACS-isolated cells and MACS MicroBead-enriched cells can all be analyzed using these reagents. The per cell price is 12.5¢ when 10,000 cells are run on one channel, and 25¢ when 5,000 cells are run on one channel.

For its single-cell assays, 10x introduced the Chromium Single Cell Controller in 2016, which has a current list price of $75,000. The Chromium Controller for both single-cell solutions and linked-read technology for genomics is $125,000. According to Brian R. Fritz, PhD, Senior Product Manager, Single Cell Genomics, the system is priced to be attractive to core labs. Using the Controller, 100 to more than 10,000 cells can be partitioned per channel in less than 7 minutes; 1–8 channels can be run in parallel. Key to meaningful single-cell analysis is the low doublet rate of only 0.9% per 1,000 cells.

The Chromium Single Cell V(D)J Solution workflow is fast: Cells are partitioned and bar-coded in less than 7 minutes. Reverse transcription takes 1 hour and then the encapsulated cells are disrupted. 5’-barcoding eliminates the bias that can come from multiplexed PCR, and enables the detection of germline and somatic variants across the entire V(D)J segment. A UMI (Unique Molecular Identifier) enables quantification. NGS library preparation takes about eight hours, and then the samples are ready for sequencing on an Illumina HiSeq 4000/2500, NextSeq or MiSeq sequencer.

The comprehensive solution also includes a complete software suite for the rapid analysis and visualization of large V(D)J datasets, designed for researchers without bioinformatics expertise. The Cell Ranger pipeline leverages barcoding to perform gene expression analysis with scalable single-cell resolution. The Loupe for Cells visualization application features powerful but easy-to-use clustering and differential expression analysis.

Also for the single-cell sequencing market, Illumina and Bio-Rad Laboratories promoted their recently introduced joint product, the Illumina Bio-Rad Single Cell Sequencing Solution. Designed to create a robust, scalable and user friendly workflow, the system was co-developed by Bio-Rad with Illumina, leveraging Bio-Rad’s expertise in Droplet Digital technology paired with the Illumina SureCell WTA 3’ Library Prep Kit. Up to 4 biological samples can be processed in less than 5 minutes.

The solution includes the ddSEQ Single-Cell Isolator, manufactured and shipped by Bio-Rad. The ddSEQ Single-Cell Isolator can encapsulate hundreds to thousands of cells per sample. Stable and uniform droplets are generated for robust cell lysis, combined with efficient barcoding and first strand cDNA synthesis. Second strand synthesis is performed, followed by library preparation without shearing or pre-amplification, using Illumina’s Nextera tagmentation technology. The total workflow from cell encapsulation to sequencing is about two working days. Prepared single-cell libraries can be loaded directly onto an Illumina MiSeq, NextSeq or HiSeq Series System for sequencing. Data analysis is streamlined and simplified using Illumina’s single-cell-analysis BaseSpace App.

According to Leanne Huysentruyt, PhD, Digital Biology Product Manager, Americas, for Bio-Rad, the co-developed system was launched on February 10. The ddSEQ Single-Cell Isolator is priced at approximately $60,000. The list price for the 2-cartridge SureCell WTA 3’ Library Prep Kit is $2,500; the 6-cartridge kit lists for $7,200. The system directly competes with the 10x Chromium System, but there are some differences in workflow and scale; the Illumina Bio-Rad single-cell solution is designed for “smaller-scale” single-cell experiments that range from hundreds to thousands of cells from each biological specimen.

The show was also a debut for Beckman Coulter Life Sciences’ CytoFLEX LX flow cytometer, which began shipping in February 2017. The latest in the company’s series of benchtop CytoFLEX systems, it can measure up to 23 parameters, the highest number among the CytoFLEX series When the system is shipped, all 6 lasers are installed. The customer can “customize” which lasers are activated. If, at a later time, the customer wants additional capacity, then the laser can be activated using system-specific software.

The system’s use of avalanche photondiodes, not PMTs, to detect the light enables better sensitivity, according to Neil Kayal, PhD, Account Sales Consultant, US North Atlantic Region, Research Flow Cytometry. He told IBO that the use of highly multiplexed flow cytometry is of great interest in the fields of immunology and immuno-oncology. Beckman Coulter expects this system to be especially useful to researchers in pharma/biotech since so many biomarkers can be screened for simultaneously. With all 6 lasers activated (21 colors), the system price is $350,000. With 5 lasers activated (19 colors), the price is $300,000.

Bio-Rad Laboratories’ ZE5 Cell Analyzer was on display at AACR. The product is scheduled to begin shipping on April 15, priced at $550,000 fully loaded. About 20 systems were placed in 2016 during an early adopter program. The ZE5 is Bio-Rad’s first flow analyzer. As Matthew Goff, Senior Cell Biology Systems Specialist, told IBO, the company got into flow cytometry because they saw it as an upstream need to support its investment in Droplet Digital technology. The system features solid state lasers, on-board fluidics and QC beads, and is an open platform. The universal loader can handle 96-well plates, 384-well plates, single tubes or a rack of tubes. A 96-well plate, 10 µl sample/well run takes 10 minutes. According to Bio-Rad, the ZE5 is designed for flow core lab users. Bio-Rad started with a punch list of what really bothered power users of flow cytometry and then designed an instrument that solved them. Target markets also include pharma/biotech labs as well as pharma ADME/Tox labs.

MilliporeSigma previewed its SMCxPRO platform and kits at the show. The product is the result of MilliporeSigma’s acquisition of exclusive rights to Singulex’s SMC (Single Molecule Counting) technology two years ago. Compared to other digital counting systems, such as the Quanterix Simoa HD-1 Analyzer, the SMCxPRO is a compact benchtop system, according to Danielle Pepin, Product Manager, Immunoassay Protein Solutions. The SMC technology is ideal for protein targets that occur at very low levels. The current range of catalog assays is focused on cytokines, key growth factors and specific neuroassays. Researchers can develop their own assays using SMC immunoassay and optimization kits, or work with the MilliporeSigma custom services team to develop bespoke SMC immunoassay kits. The system is scheduled to be released in the second half of the year at a lower price than Singulex’s Erenna Immunoassay System.

Next year’s AACR meeting will be held April 14–18 in Chicago, Illinois.

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