Lawrence Berkeley National Laboratory

Single cell genomics, the amplification and sequencing of genomes from single cells, can provide a glimpse into the genetic make-up and thus life style of the vast majority of uncultured microbial cells, making it an immensely powerful and increasingly popular tool. This is accomplished by use of multiple displacement amplification (MDA), which can generate billions of copies of a single bacterial genome producing microgram-range DNA required for shotgun sequencing. Here, we would like to address several challenges inherent in such a sensitive method and propose solutions for the improved recovery of single cell genomes. While DNA-free reagents for the amplification of a single cell genome are a prerequisite for successful single cell sequencing and analysis, DNA contamination has been detected in various reagents, which poses a considerable challenge. Our study demonstrates the effect of UV radiation in efficient elimination of exogenous contaminant DNA found in MDA reagents, while maintaining Phi29 activity. Second, MDA is subject to amplification bias, resulting in uneven and sometimes insufficient sequence coverage across the genome. In a post-amplification method, we employed a normalization step within 454 Titanium library construction in which populations of highly abundant sequences were specifically targeted and degraded from the library via duplex-specific nuclease, resulting in decreased variability in genome coverage. While additional challenges in single cell genomics remain to be resolved, the two proposed methodologies are relatively quick and simple and we believe that their application will be of high value for future single cell sequencing projects.