New Method to Boost Single Cell Genomics

Scientists can now get a more accurate look at a single cell’s genome, thanks to a new technique. Since the procedure captures more of a cell’s genetic code than methods past, researchers can better resolve small genetic changes in individual cells that drive such processes as evolution, cancer, and maybe even neurodegenerative disease. Led by Sunney Xie, researchers from Harvard University reported their method in the December 21 issue of Science. They used it to identify single nucleotide and gene copy number variations among cells in the same line. Both types of variants can cause Alzheimer's, Parkinson's, and other neurological diseases.

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.

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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

Comments

  1. Researchers began publishing the sequences of single cell genomes back in 2006 (Zhang et al., 2006), and the technology has continued to evolve until today (see Fan et al., 2010; Navin et al., 2011; Gundry et al., 2012; Hou et al., 2012; Wang et al., 2012). The improvement here lies in getting a smooth representation of the original DNA regions of the genome. Their method reduces variation and bias. This is very good for determining copy number variation. But another key component of accuracy (that the authors do not mention) is haplotype phase, which tells you if you have 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. A combination of the MALBAC and long fragment read (LFR) methods would be ideal. Although previous work required more than one cell (Peters et al., 2012), note that here, Zong et al. also explicitly state, "our strategy to reduce the false positive rate was to sequence two or three kindred cells derived from the same cell."

    References:

    Zhang K, Martiny AC, Reppas NB, Barry KW, Malek J, Chisholm SW, Church GM. Sequencing genomes from single cells by polymerase cloning. Nat Biotechnol. 2006 Jun;24(6):680-6. PubMed.

    Fan HC, Wang J, Potanina A, Quake SR. Whole-genome molecular haplotyping of single cells. Nat Biotechnol. 2011 Jan;29(1):51-7. PubMed.

    Navin N, Kendall J, Troge J, Andrews P, Rodgers L, McIndoo J, Cook K, Stepansky A, Levy D, Esposito D, Muthuswamy L, Krasnitz A, McCombie WR, Hicks J, Wigler M. Tumour evolution inferred by single-cell sequencing. Nature. 2011 Apr 7;472(7341):90-4. PubMed.

    Gundry M, Li W, Maqbool SB, Vijg J. Direct, genome-wide assessment of DNA mutations in single cells. Nucleic Acids Res. 2012 Mar;40(5):2032-40. PubMed.

    Hou Y, Song L, Zhu P, Zhang B, Tao Y, Xu X, Li F, Wu K, Liang J, Shao D, Wu H, Ye X, Ye C, Wu R, Jian M, Chen Y, Xie W, Zhang R, Chen L, Liu X, Yao X, Zheng H, Yu C, Li Q, Gong Z, Mao M, Yang X, Yang L, Li J, Wang W, Lu Z, Gu N, Laurie G, Bolund L, Kristiansen K, Wang J, Yang H, Li Y, Zhang X. Single-cell exome sequencing and monoclonal evolution of a JAK2-negative myeloproliferative neoplasm. Cell. 2012 Mar 2;148(5):873-85. PubMed.

    Wang J, Fan HC, Behr B, Quake SR. Genome-wide single-cell analysis of recombination activity and de novo mutation rates in human sperm. Cell. 2012 Jul 20;150(2):402-12. PubMed.

    Peters BA, Kermani BG, Sparks AB, Alferov O, Hong P, Alexeev A, Jiang Y, Dahl F, Tang YT, Haas J, Robasky K, Zaranek AW, Lee JH, Ball MP, Peterson JE, Perazich H, Yeung G, Liu J, Chen L, Kennemer MI, Pothuraju K, Konvicka K, Tsoupko-Sitnikov M, Pant KP, Ebert JC, Nilsen GB, Baccash J, Halpern AL, Church GM, Drmanac R. Accurate whole-genome sequencing and haplotyping from 10 to 20 human cells. Nature. 2012 Jul 12;487(7406):190-5. PubMed.

    View all comments by George Church

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Papers

  1. Fan HC, Wang J, Potanina A, Quake SR. Whole-genome molecular haplotyping of single cells. Nat Biotechnol. 2011 Jan;29(1):51-7. PubMed.
  2. Navin N, Kendall J, Troge J, Andrews P, Rodgers L, McIndoo J, Cook K, Stepansky A, Levy D, Esposito D, Muthuswamy L, Krasnitz A, McCombie WR, Hicks J, Wigler M. Tumour evolution inferred by single-cell sequencing. Nature. 2011 Apr 7;472(7341):90-4. PubMed.
  3. Wang J, Fan HC, Behr B, Quake SR. Genome-wide single-cell analysis of recombination activity and de novo mutation rates in human sperm. Cell. 2012 Jul 20;150(2):402-12. PubMed.
  4. Gundry M, Li W, Maqbool SB, Vijg J. Direct, genome-wide assessment of DNA mutations in single cells. Nucleic Acids Res. 2012 Mar;40(5):2032-40. PubMed.

Primary Papers

  1. 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 Dec 21;338(6114):1622-6. PubMed.

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