Xi Larry, Belyaev Alexander, Spurgeon Sandra, Wang Xiaohui, Gong Haibiao, Aboukhalil Robert, Fekete Richard
Fluidigm Corporation, South San Francisco, California, United States of America.
PLoS One. 2017 Jul 19;12(7):e0181163. doi: 10.1371/journal.pone.0181163. eCollection 2017.
A central challenge in sequencing single-cell genomes is the accurate determination of point mutations, phasing of these mutations, and identifying copy number variations with few assumptions. Ideally, this is accomplished under as low sequencing coverage as possible. Here we report our attempt to meet these goals with a novel library construction and library amplification methodology. In our approach, single-cell genomic DNA is first fragmented with saturated transposition to make a primary library that uniformly covers the whole genome by short fragments. The library is then amplified by a carefully optimized PCR protocol in a uniform and synchronized fashion for next-generation sequencing. Each step of the protocol can be quantitatively characterized. Our shallow sequencing data show that the library is tightly distributed and is useful for the determination of copy number variations.
对单细胞基因组进行测序的一个核心挑战是准确确定点突变、这些突变的相位，以及在极少假设的情况下识别拷贝数变异。理想情况下，这要在尽可能低的测序覆盖度下完成。在此，我们报告了我们尝试通过一种新型文库构建和文库扩增方法来实现这些目标的情况。在我们的方法中，单细胞基因组DNA首先通过饱和转座进行片段化，以构建一个由短片段均匀覆盖整个基因组的初级文库。然后，通过精心优化的PCR方案以均匀且同步的方式对该文库进行扩增，用于下一代测序。该方案的每一步都可以进行定量表征。我们的浅测序数据表明，该文库分布紧密，可用于拷贝数变异的测定。
