Single-cell genome and transcriptome sequencing methods are generating a fresh wave of biological insights into development, cancer and neuroscience. Kelly Rae Chi reports in Nature Methods.
As reported in the article, one of several groups applying single-cell genome sequencing to IVF, Sunney Xie at Harvard University and his collaborators have tested their new whole-genome amplification methods on the first and second polar bodies, small cellular castoffs of the fertilized donor egg that reflect its chromosomal health. In a recent paper, Xie’s team showed that in eight female donors, polar-body biopsy and single-cell sequencing could correctly infer both embryo aneuploidy—too many chromosomes, as in the case of Down’s syndrome, or too few—and single-nucleotide variations inherited from either parent. Detecting aneuploidy may require sequencing as little as one out of every hundred genomic regions on average, making the strategy cheaper and more accurate than traditional methods, Xie says.
Xie and his collaborators on the paper, Fuchou Tang of Peking University and Jie Qiao of Peking University Third Hospital, have launched a clinical study of women undergoing IVF. The team will amplify and sequence whole genomes of the polar bodies of participants’ embryos to see whether they are fit for transfer. Such a step toward the clinic seemed impossible only 2 years ago, says Xie, adding that people desperate to have a baby free of a devastating genetic disorder have been e-mailing him. The study’s first baby could be born within the year. “I didn’t anticipate that [our technique] would be used so quickly for patients,” he says.
Single-cell sequencing
Single-cell sequencing is no small feat. The amount of DNA or RNA in a single cell starts at a few picograms—not even close to the quantity that today’s sequencing machines demand. So scientists must amplify these molecules and do so in ways that minimize technical errors while surveying sequences as broadly and evenly as possible. Until recently, many researchers doubted that sequencing of single cells could be reliably conducted by any but a few experts.
Although a handful of groups sowed the seeds for single-cell genome and transcriptome sequencing approaches years ago, the methods have more recently started to make their way to the masses, and a community has formed around their application in areas including neuroscience, cancer and microbial ecology. “Almost since the first day that PCR was invented, people began trying to use it to do single-cell gene expression and genome analysis,” says Stephen Quake at Stanford University, cofounder of Fluidigm. “But [single-cell sequencing] really is just taking off for a bunch of reasons.”
From a few molecules of RNA
Sequencing a cell’s transcriptome hinges on the ability to amplify large amounts of the complementary DNA (cDNA) that is synthesized from RNA. Capturing small amounts of RNA as cDNA and amplifying the cDNA extensively are difficult to do evenly and efficiently.
In 1990, transcriptome analysis at the resolution of single cells was made possible by Norman Iscove’s group, who amplified cDNAs exponentially using PCR. In the early 1990s, Eberwine and his colleagues came up with a technique that generated cDNA from single live neurons and performed linear amplification by transcribing RNA from the cDNA. With the advent of microarrays, scientists used both linear and exponential amplification strategies to identify differences in gene expression among single cells.
High-throughput RNA sequencing (RNA-seq) came onto the scene in 2008, and shortly after, researchers coupled it to such amplification techniques to get a more detailed look at single-cell transcriptomes. For a 2009 study, Tang, then working in M. Azim Surani’s laboratory at the Gurdon Institute at the University of Cambridge, showed that it was possible to detect—from a single mouse blastomere—the expression of thousands more genes than had been revealed using microarrays .
Amplifying the genome
Developing a way to amplify whole genomes of single cells took a bit longer because only one or two unique copies of DNA exist in the cell. The method lagged behind RNA amplification until 2005, when Roger Lasken’s group became the first to amplify and sequence DNA from a single cell, that of an Escherichia coli bacterium, using the multiple displacement amplification (MDA) method that they had developed. That sparked a vigorous effort by microbiologists to generate reference genomes for diverse, uncultivable bacterial species.
The cellular patchwork of cancer
From prognostics to disease monitoring, cancer research stands to benefit enormously from single-cell sequencing approaches. Cancer cells often undergo high mutation rates, and tumors tend to be heterogeneous. Identifying which subsets of cells, called clones, are present and evolve into metastases or respond in a certain way to chemotherapy is critical to understanding and fighting the disease. In particular, circulating tumor cells (CTCs)—which break off from a tumor and seed a cancer’s metastasis—are those rare cells whose genomes or transcriptomes might offer clues for diagnosis, monitoring or treatment.
Reference:
Singled out for sequencing. Nature Methods. 2014;11:13-17
mRNA-Seq whole-transcriptome analysis of a single cell. Nature Methods.2009;6:377-382