Why one person has a different number of copies of a certain gene than another person is not understood, but the implications of such copy number variation are increasingly appreciated. Now that sequencing technology has made it possible to measure copy number variation (CNV) from cell samples, and even single cells, researchers are finding that an abnormal copy number of some genes are associated with cancer and other complex diseases. Here are some technologies that enable single-cell CNV detection to be used to fight diseases.
CNV detection tools
Amplifying the DNA from a single cell is needed prior to detecting and measuring copy numbers of genes. Takara Bio’s PicoPLEX WGA family of products offers whole genome amplification (WGA) technology suitable for single-cell CNV detection. “It’s optimized for unbiased amplification of single copy genomic DNA from low input samples” such as single cells, says Kajal Choudhary, senior manager of corporate development in reproductive health strategy at Takara Bio. “Measuring all the DNA information from a small amount is very difficult, so whole genome amplification technology is an essential step in library preparation for high-throughput sequencing.”
An increasing number of researchers are measuring both copy number variations and gene expression. “For example, there is a clear correlation between cancer and copy number variation and its impact on gene expression,” says Suvarna Gandlur, associate director of NGS product management and marketing at Takara Bio. The PicoPlex WGA technology is also used to measure “copy number variation in tumors and cancer cells to understand progression or disease state, and to tie it to gene expression,” she says.
Another application of Takara Bio’s WGA single cell products is pre-implantation genetic testing (PGT) of embryos. “With PGT, single-cell analysis can give more information about the nature of aneuploidy or the nature of genetic abnormalities,” says Choudhary. Looking forward, this technology may enable PGT testing on cell-free DNA from culture media, instead of using DNA from cells removed from an embryo. “We can also use our whole genome amplification technology to amplify cell-free DNA for non-invasive PGT testing,” says Choudhary. “We’re investing in and optimizing a workflow for this, but a lot of clinical validation and testing needs to be done before it becomes widely adopted.”
Quality sample preparation is important for high-quality results in CNV detection. Samuel Rulli, associate director of global product management for NGS at QIAGEN, notes that variability in single-cell sequencing results can originate from early processing steps. “Variability in results can be traced back to how the cell has been isolated, stored, and even shipped from a collection point to its final destination,” he says. “The Repli-g Advanced DNA Single Cell kit enables high uniformity in DNA amplification because it includes a cryo-protect reagent to minimize DNA damage during single-cell capture, storage, or shipping.”
QIAGEN’s Repli-g Advanced DNA Single Cell kit uses multi-displacement amplification of cellular DNA in a simple protocol that takes only 2 hr 20 min. The QIAseq FX Single Cell DNA library kits are available for next-generation sequencing, and include unbiased enzymatic fragmentation for NGS library construction. “The workflow often involves capturing a single cell by picking, sorting or enrichment, stabilizing the cell if it is going to be analyzed at a later time, lysing the cell, amplifications of the DNA, and then downstream analysis by digital PCR or next-generation sequencing,” says Rulli.
A whole genome platform for CNV detection
Single-cell DNA sequencing can reveal genomic heterogeneity that is otherwise missed when studying groups of cells. Cancer and other diseases are associated with genomic heterogeneity that arises from the development of subpopulations of cells, or clones. 10x Genomics’s Chromium Single-Cell CNV Solution helps researchers identify genomic heterogeneity and map clonal evolution. “[It uses] our technology for generating cell beads and gel beads, and enables preparation of hundreds to thousands of single cells for CNV profiling within minutes on either the Chromium Controller or the Chromium Single-Cell Controller,” says Michael Schnall-Levin, senior VP of R&D and founding scientist at 10x Genomics.
The typical workflow for 10x Genomics begins with a suspension of single cells, and ends in a library ready for next-generation sequencing. Applications of their platform include evaluating tumor heterogeneity, clonal evolution, neuronal mosaicism, and genetic and metabolic disorders. “Our customers have been using CNV analysis in a variety of research areas including oncology, neuroscience, human genetics, and stem cell biology,” says Schnall-Levin. “It also allows for further examination of the clonal structure of cancer samples, including spatial distribution across tumor slices and longitudinal changes.”
A targeted platform for CNV detection
In contrast to the whole-genome approach, Mission Bio’s Tapestri platform uses a targeted approach to detect both point mutations and copy number variation from individual cells using genes of interest. Like 10x Genomics and others, Mission Bio uses a microfluidics droplet-based technology, and uses it to analyze 10,000 cells at once. Each droplet encapsulates one cell, serving as a space where chemical reactions occur. A unique cell barcode marks each droplet for subsequent identification. Mission Bio’s technology is unique in that it separates the harsh reactions needed to lyse cells from the more chemically sensitive DNA amplification reactions. This enables the platform to measure DNA accurately and reproducibly with single-cell resolution.
Using targeted panels to analyze many genomic regions, the Tapestri system establishes the baseline level of amplification for normal cells. Through comparison to this baseline, the technology can reveal variations in copy number. “We can design panels across entire chromosomal arms to look at aneuploidy, or down to a single specific gene to determine copy gain or loss and, in the same workflow, identity point mutations,” says Anup Parikh, VP of software and informatics at Mission Bio.
Researchers use Mission Bio’s Tapestri platform to understand the evolution of different types of cancers driven by copy number changes, point mutations, or a combination of both. “Sometimes it’s the co-occurrence of point mutations, with loss of heterozygosity, or copy gains that drive it to be aggressive or resistant to treatment,” says Parikh. Translational research on cancer patient biopsies using the Tapestri platform illustrates how mutations and copy number changes can drive the progression of cancer, which bears on the development and delivery of targeted treatments. “One clone might be driven by a mutation and a copy number variation, while three different co-occurring mutations drive another,” he says. “Identifying the different clones can help tailor the right combo therapy rather than just one treatment.”
Mission Bio’s translational focus has taken them to major cancer centers—such as MD Anderson, Memorial Sloan Kettering, and UCSF—where researchers are working to understand the heterogeneity of cancer. “CNV is oftentimes an early driver in cancer evolution and indicative of the severity of the cancer,” says Parikh. “We see simultaneous mutation and CNV detection at single-cell resolution as a fundamental tool for better understanding the complexity of cancer, so that we can develop better therapies faster and more successfully.”