PubMed Highlights: an improved strategy for Whole-Genome Sequencing of Single Cells

MALBAC single-cell whole-genome amplification, Science 21-12-2012: vol. 338 no. 6114 1622-1626.

Scientists of Harvard University developed a new technique they call MALBAC (multiple annealing and looping-based amplification cycles) that yields better sequence coverage than has previously been available for single-cell genome sequencing. Sequencing MALBAC-amplified DNA achieves 93% genome coverage ≥1x for a single human cell at 25x mean sequencing depth, as reported in the latest issue of Science.
In MALBAC, genomic DNA is copied to form looped products that 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 PCR or multiple displacement amplification. After five MALBAC cycles, the DNA loops are collected and used as templates for further amplification by PCR. Apparently, the linear amplification step reduces the sequencing bias. “Most of the amplification bias is generated in the first few cycles of PCR,” one of the authors of the study says. “By doing linear amplification first we avoid this strong bias. That makes it very even across the genome.”

MALBAC was successfully used for this two impressive studies:

Science. 2012 Dec 21;338(6114):1622-6. doi: 10.1126/science.1229164.

Genome-wide detection of single-nucleotide and copy-number variations of a single human cell.

Zong C, Lu S, Chapman AR, Xie XS.

Source

Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.

Abstract

Kindred cells can have different genomes because of dynamic changes in DNA. Single-cell sequencing is needed to characterize these genomic differences but has been hindered by whole-genome amplification bias, resulting in low genome coverage. Here, we report on a new amplification method-multiple annealing and looping-based amplification cycles (MALBAC)-that offers high uniformity across the genome. Sequencing MALBAC-amplified DNA achieves 93% genome coverage ≥1x for a single human cell at 25x mean sequencing depth. We detected digitized copy-numbervariations (CNVs) of a single cancer cell. By sequencing three kindred cells, we were able to identify individual single-nucleotide variations (SNVs), with no false positives detected. We directly measured the genome-wide mutation rate of a cancer cell line and found that purine-pyrimidine exchanges occurred unusually frequently among the newly acquired SNVs.

PMID: 23258894

Science. 2012 Dec 21;338(6114):1627-30. doi: 10.1126/science.1229112.

Probing meiotic recombination and aneuploidy of single sperm cells by whole-genome sequencing.

Lu S, Zong C, Fan W, Yang M, Li J, Chapman AR, Zhu P, Hu X, Xu L, Yan L, Bai F, Qiao J, Tang F, Li R, Xie XS.

Source

Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.

Abstract

Meiotic recombination creates genetic diversity and ensures segregation of homologous chromosomes. Previous population analyses yielded results averaged among individuals and affected by evolutionary pressures. We sequenced 99 sperm from an Asian male by using the newly developed amplification method-multiple annealing and looping-based amplification cycles-to phase the personal genome and map recombination events at high resolution, which are nonuniformly distributed across the genome in the absence of selection pressure. The paucity of recombination near transcription start sites observed in individual sperm indicates that such a phenomenon is intrinsic to the molecular mechanism of meiosis. Interestingly, a decreased crossover frequency combined with an increase of autosomal aneuploidy is observable on a global per-sperm basis.

PMID: 23258895