To generate enough DNA for sequencing, a single cell’s genome has to be amplified many times over. This can introduce errors. Some regions are easier to copy than others, for example, and can dominate a sample. This is especially true when researchers rely on the polymerase chain reaction (PCR), because it preferentially and disproportionately amplifies easily copied regions.

First author Chenghang Zong and colleagues developed a more conservative method, called multiple annealing and looping-based amplification cycles (MALBAC). It amplifies genomic DNA cautiously at first. Instead of using every DNA fragment as a template in multiple rounds of amplification, as does PCR, MALBAC loops any duplicated DNA from the first round, leaving only the original DNA as linear pieces for the next amplification step. That limits imbalances. After five cycles of MALBAC, PCR takes over to do the rest.

A new method to amplify a single cell’s DNA for sequencing reveals copy number variations (green arrows) among cells of the same line. Image courtesy of Science/AAAS

Researchers found that MALBAC covers up to 93 percent of a single human cell’s genome. With such high capture, the group easily detected copy number variations in single cells and spotted single nucleotide variations between related cells. Another key component of accuracy is haplotype phase. This tells the scientist if there are two mutations affecting both copies of a gene, or those same two mutations in only one copy, leaving the other copy unaffected. "Knowing if you have one good copy or zero is a big deal for clinical (and research) accuracy," noted George Church, Harvard Medical School, in an e-mail to Alzforum (see full comment below).—Gwyneth Dickey Zakaib.

Reference:
Zong C, Lu S, Chapman AR, Xie XS. Genome-Wide Detection of Single-Nucleotide and Copy-Number Variations of a Single Human Cell. Science 2012 December 21;(380):1622-1626. Abstract