Massively Parallel Polymerase Cloning and Sequencing of Single Cells

Genome sequencing of single cells has a variety of applications including characterizing difficult-to-culture microorganisms in various environments and human body sites, and identification of somatic mutations in single tumor cells. A key step is to generate clonal copies of DNA molecules within a single cell by DNA polymerase, a process that we called polymerase cloning. Polymerase cloning has been implemented through a number of amplification strategies, including multiple displacement amplification (MDA), hyper-branched rolling circle amplification (hRCA) and PCR based approaches. A common limitation is the highly biased representation of the template genome after amplification, which increases the sequencing cost, limits the genome coverage and compromises the sensitivity of detecting mutations.

To tackle this challenge, we have developed a massively parallel polymerase cloning method using microwell arrays. In this method, hundreds to thousands of single cells are randomly deposited into a PDMS microwell array fabricated on a standard microscope slide. Polymerase cloning of all the individual cells is performed in parallel in a nanoliter volume, and the amplification is monitored in real-time with an epifluorescent microscope. Positive polymerase clones are extracted with a micromanipulator, and converted into multiplexed sequencing libraries for Illumina sequencing. This method is inherently scalable, low-cost and yields an unbiased representation of the genome from single cells. Results on de novo genome sequencing of single environmental microbes and resequencing of single mammalian cells will be presented.