Single Cell Sequencing: The Technology, Challenge and Future (IV)

Wolf Reik hopes that epigenetics technology can also reach the level of single cell detection as soon as possible.

What is more difficult than genome and transcriptome research is the epigenome study that attaches to the genome in the form of chemical markers and regulates gene expression. Although the current epigenetics technology has not yet reached the level of single-cell research (because traditional epigenetics research techniques will degrade DNA), researchers are still eager to see the epigenome of individual tumor cells. Tang’s research team has developed a new technology that can study the modification of DNA methylation within a single-cell genome (Genome Res. 23, 2126–2135, 2013). Tang believes that single-cell technology is also really required for epigenome research. Only in this way can researchers understand the difference between this tumor cell and the surrounding tumor cells, and this difference is caused by methylation modification. It is also caused by other mechanisms. The Wolf Reik team at the Wellcome Trust Sanger Institute in the UK analyzed the methylome of 50 to 100 cells, and he said he really wanted to go one step further.

2.6 Exploration to neuron cells

Neuron cells are the latest object used for single-cell research, and scientists are actually not quite sure what information and conclusions can be obtained through these studies. It was only recently that there was experimental evidence that neurons also have different genomes. Despite these research results, scientists are still confused about the diversity of neuronal cells. As early as 2001, Jerold Chun, who was still working at the University of California, San Diego, discovered chromosomal aneuploidy in the brain of mice, and then in human brain cells in 2005. The same phenomenon was found. According to McConnell, who was a graduate student in the Chun laboratory at that time, after getting these results, no one knew what to do next. They are equivalent to discovering the tip of the iceberg. If there is aneuploidy in the cell, there must be a lot of gene mutations, or genome mutations.

Almost at the same time, another group of researchers found that in the human genome, on average, each genome contains 80 to 100 potentially viable L1 elements (this is a kind of self-replication and self-pasting in the entire genome DNA elements), and in brain neuron cells, these L1 elements are active. This study, as well as some other research results, have proved that the genome is at least possible to have diversity, but no one can say clearly how great this variation is.

According to Thomas Insel of the US National Institute of Mental Health, they are just beginning to try to understand the molecular diversity of brain cells. The single-cell research technology in this field plays a key role, not only in determining the (classification) type of neuronal cells and glial cells, but also in helping us understand the experience and development of a certain area of the brain What is the role of gene expression.

Scientists can use several methods to detect single-cell genome variation. The Christopher Walsh team of Harvard Medical School conducted a single-cell L1 element insertion study on 300 neurons taken from the dead brain (Cell 151, 483–496, 2012). They only found a few L1 insertion elements, which indicates that L1 elements should not be the main cause of genomic diversity, but at least in cerebral cortex cells and caudate nucleus cells.

In 2013, several other research groups also conducted genome-wide scanning studies on single human neuronal cells. For example, in an article published in November 2013, a genome-wide sequencing study was performed on 110 frontal cortex neuron cells in the brains of three healthy people. The results were quite surprising. Large segments of CNV mutations (Science 342, 632–637, 2013). Studies on neuronal cells derived from healthy human skin cells have also found the same situation, and these neuronal cells have more CNV than skin cells from which they are derived, which shows that this neuron derived from iPS cells Cells are a very good research material, suitable for research work on cell diversity.

In fact, despite these discoveries, neuroscientists still have a headache because they do not know what these somatic mutations mean. Ira Hall, a geneticist at the University of Virginia, is also one of the collaborators of this article published in Science. He believes that these studies mean that the brain ’s resistance to influence and interference is very weak. In addition, genomic mosaicism can also affect people ’s risk of developing tumors and other diseases. To find out which parts of the brain are more susceptible to interference than other parts and how different the different parts of the brain are, researchers have to study more cells before they can find the answer. McConnell, who is currently engaged in research in this area, believes that he still knows nothing.

2.7 The further development

Although single-cell technology has the potential to solve many major problems in the life sciences, technological progress is far from over. For example, researchers must study how to distinguish true biological differences from the background noise of the test technology itself. Joakim Lundeberg of KTH Royal Institute of Technology in Sweden (who has developed tissue RNA sequencing technology in their laboratory) believes that single cell RNA and DNA sequencing technology is far from being powerful enough, he said that they also need to analyze more single cells in one experiment in order to solve the problem of background noise, which can at least deepen their understanding of the differences between different cells in the same tissue.

Due to various problems, such as cell separation, data calculation, and specificity issues when used in different fields, etc., Blainey hopes that single cell research technology can make greater progress in the next few years.

For newcomers to this field, which transcriptome sequencing technology they choose may be enough for them to have a headache for a long time. Regarding this issue, it should depend on the purpose of the research, such as whether you want to analyze multiple cells to find homologous transcripts, or you want to find low-abundance RNA. “But it’s always a good thing to have multiple methods to choose from,” Quake said. Quake ’s team found that if the reaction volume during pretreatment is controlled to be upgraded (they use the C1 system provided by Fluidigm), then the detection effect of single-cell qPCR technology and single-cell RNA sequencing technology is almost the same (Nat. Methods 11, 41–46, 2014).

With the introduction of commercial products, and the various laboratories who have summed up their “unique secrets” after years of practice, the choice of genome amplification technology is also improving. However, because everyone uses different techniques for genome amplification, it is difficult to directly compare different research results.

At the same time, researchers engaged in cancer research, brain neuroscience research, microbiological research, and drug development and other fields of research will also benefit from these technological advancements. And these technological advancements will also attract many outstanding talents to join the field of single-cell research, such as Reik, who has already made a lot of achievements in epigenetics research. Reik only participated in the single-cell academic conference for the first time last year, and has never been exposed to single-cell research before. Reik is very excited to see so many new technologies. He pointed out that at the beginning people will be excited by the technology itself, and it will not be long before people will use these new technologies to solve important life science problems, which will be more exciting.

To be continued in Part V…