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Sequencing from single cells

December 19, 2012 This article courtesy of Nature News.

Technique finds more mutations within individual cells.

Sequencing projects are usually a mishmash. To get enough DNA, thousands or even millions of cells are required. Knowing which mutations are in which cells is impossible. So is detecting mutations in rare cells. A technique reported today makes it easier to find genetic differences across individual cells. Such differences may help explain how cancer becomes more malignant, how reproductive cells emerge, and even how individual neurons differ.

Sequencing an individual cell requires making many copies of its DNA. But some portions of the genome are copied much more extensively than others, a problem known as amplification bias. The least-copied regions of the genome are undetectable among the most-copied regions, and so most attempts at single-cell sequencing cover at best around 70% of the genome. Rates of around 40% are typical.

Sunney Xie at Harvard University and colleagues have now described a new way to copy DNA that covers over 90% of the genome. The technique is called MALBAC, which stands for multiple annealing and looping-based amplification cycles. After DNA from a single cell is purified, short DNA molecules called primers are added. These primer consist of 8 nucleotides that vary from primer to primer plus a common sequence of 27 nucleotides.

The variable portions bind randomly across the genome, allowing DNA replication to begin. The common 27-nucleotides sequence is incorporated into the copied strands, so that twice-replicated strands loop up on themselves. That looping prevents further copying and reduces amplification bias.

“MALBAC opens a door to many critical questions,” says Bing Ren, who studies gene regulation at the University of California, San Diego. For example, it can examine copy number variation and chromosomal translocations across a population of cells. It also detects variants across a larger portion of the genome.

Though working out the precise conditions was difficult, other researchers should be able to use MALBAC without investing in expensive equipment, says Xie.

“Once people have the recipe, it is easy to reproduce.” “I think people are going to start using it right away,” says James Eberwine, who works on single-cell genetics at the University of Pennsylvania Medical School. He adds that researchers may have to tweak conditions – such as the ratio of primers to genomic DNA to get experiments to work.

But while MALBAC covers the genome more thoroughly than other techniques, it is still not perfect. It still misses perhaps a third of single nucleotide variants. Also, the enzyme that copies DNA is itself error prone, so the copying process itself introduces variants that do not exist in individual cells.

Xie was able to weed out all false positives, but doing so required comparing individually sequenced genomes from three closely related cells. That will increase costs, and could prove impossible for certain cell types, says Nicholas Navin at the MD Anderson Cancer Center, who has developed his own techniques for single-cell sequencing. However, when researchers need to sample the most DNA from a single cell, they are likely to turn to MALBAC.

“This paper will have a big impact on the single-cell nucleic acid analysis field,” says Paul Blainey, who develops single-cell analysis techniques at the Massachusetts Institute of Technology.

Zong, C., Lu, S., Chapman, A.R. and Xie, X.S. Science 338, 1622-1626

ALTERNATE LEDE Researchers have sequenced humans, strawberries, honey bees, chickens, rats, frogs, and more. More challenging than sequencing any individual species, however, is sequencing an individual cell. A technique reported in Science today can detect many small mutations from single cells.

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