Using a newly developed single-cell sequencing technique, a team of scientists has decoded the DNA of individual sperm cells as well as individual colon cancer cells. The results provide new insights into genetic recombination, male infertility, and cancer.
"The looping of amplicons prevents them from being further amplified in later cycles of MALBAC, until amplification by PCR," said study author Sunney Xie, a chemist at Harvard University. Along with Harvard post-doc Chenghang Zong, Harvard graduate student Sijia Lu, and other collaborators, Xie used the new method to sequence the genome of individual colon cancer and sperm cells and reported the results in two papers published in the December 21 issue of Science (1–2).
The team developed the new method to reduce the whole-genome amplification bias and low genome coverage of other methods. In collaboration with Ruiqiang Li’s group at Peking University, Xie’s team used MALBAC to improve the genome coverage of individual sperm cells published in previous studies that used multiple displacement amplification (MDA) for prepping DNA and microarray for genotyping (3).
The authors of the new studies argue that MALBAC has better amplification uniformity than MDA, and provides genome-wide sequence rather than focusing on selective single-nucleotide polymorphism (SNP) markers obtained from genotyping. As a result, "our personal recombination map has substantially higher resolution than previously reported and allows us to study characteristics of the recombination spots more closely," said Li.
The team used MALBAC to sequence the genome of 99 separate sperm cells from an anonymous 40-year-old donor. The technique identified recent single-nucleotide variations and copy-number variations in the DNA, which was also scanned for abnormal genome variations that could affect fertility. The results show lower recombination rates near sites where transcription starts.
In a second experiment, the team used MALBAC to sequence three cells from the same colon cancer cell lineage to measure the genome-wide mutation rate for the cell line. With the results, the scientists could begin to study the cancer cells' heterogeneity, a common feature of tumors that may contribute to drug resistance.
Overall, the results suggest that there is a lower frequency of transition mutations, where adenine changes to guanine and cytosine to thymine, than transversion mutations. Most mechanisms of DNA damage favor transitions over transversions, normally leading to a high ratio of transitions to transversions. "The fact that we observe a low ratio indicates that a less common mechanism is responsible for the mutations we find," Xie said.
1. Lu, S., C. Zong, W. Fan, M. Yang, J. Li, A. R. Chapman, P. Zhu, X. Hu, L. Xu, L. Yan, F. Bai, J. Qiao, F. Tang, R. Li, and X. S. Xie. 2012. Probing meiotic recombination and aneuploidy of single sperm cells by Whole-Genome sequencing. Science 338(6114):1627-1630.
2. Zong, C., S. Lu, A. R. Chapman, and X. S. Xie. 2012. Genome-Wide detection of Single-Nucleotide and Copy-Number variations of a single human cell. Science 338(6114):1622-1626.
3. Wang, J., H. C. Fan, B. Behr, and S. R. Quake. 2012. Genome-wide Single-Cell analysis of recombination activity and de novo mutation rates in human sperm. Cell 150(2):402-412.