A tiny genetic change in a single cell could hold the seeds of an inherited disease or heightened cancer risk. But such changes are hard to find, because existing DNA amplification methods are poorly suited for single-cell genome sequencing—they are better at bulk sequences. However, a single cell is often all that’s available for applications such as prenatal screening or analysis of circulating tumor cells.
A new amplification method smooths out sequence biases that hamper the bulk techniques to achieve uniform single-cell genome coverage. X. Sunney Xie, Chenghang Zong, Sijia Lu, and Alec R. Chapman of Harvard University developed the technique they call MALBAC, for multiple annealing and looping-based amplification cycles (Science, DOI: 10.1126/science.1229164). It yields better sequence coverage than has previously been available for single-cell genome sequencing.
In MALBAC, genomic DNA is copied to form looped products. These loops can’t serve as templates, so in each cycle only the genomic DNA can be copied. The amount of DNA increases linearly rather than exponentially as it would in other amplification methods such as polymerase chain reaction (PCR) or multiple displacement amplification. After five MALBAC cycles, Xie and coworkers collect the DNA loops and use them as templates for further amplification by PCR.
The linear amplification step reduces the sequencing bias. “Most of the amplification bias is generated in the first few cycles of PCR,” Xie says. “By doing linear amplification first we avoid this strong bias. That makes it very even across the genome.” MALBAC results in genome coverage more uniform than multiple displacement amplification, the current single-cell standard, but less uniform than bulk sequencing.
In one demonstration, the Harvard team sequenced individual genomes from cells obtained by allowing a single cancer cell to divide 20 times. They achieved 93% coverage of individual genomes and identified 35 newly acquired single-nucleotide mutations. They also determined copy number variations, replication errors that result in abnormal numbers of particular DNA sequences. Data from other amplification methods are too noisy to reliably detect copy number variations.
The Harvard scientists also collaborated with colleagues at Peking University in China to sequence the genomes of 99 individual human sperm cells using MALBAC (Science, DOI: 10.1126/science.1229112). The team was able to identify sperm with duplicated or missing chromosomes. They were also able to “phase” the genome to determine crossover points where the sequence switches from maternal to paternal origin, which is an important part of genetic recombination during meiosis. The data were of higher resolution than those from previous efforts to sequence genomes in individual sperm cells.
MALBAC “is about halfway home in terms of making single-cell genome sequencing comparable to bulk DNA sequencing in terms of the uniformity,” says Jay A. Shendure, a professor of genome sciences at the University of Washington, Seattle. Shendure plans to try the method himself. “That is perhaps the clearest indicator that we view it as a potentially important advance.”