Accurate Genome Analysis of Single Cells

Whole genome analysis can be performed by next-generation-sequencing (NGS) techniques, microarrays, or parallel real-time PCR addressing multiple genomic regions. These analyses require a minimal amount of gDNA from 100–1000 ng, equivalent to 16,000–160,000 cells. For analysis of genomic differences between individual cells, accurate replication of the single-cell genome is required. Here, we describe the reliability of single-cell whole genome amplification (WGA) and its application in NGS and real-time PCR. Single cells were obtained by picking cells under the microscope or by dilution. Amplification was performed for 8 hours at 30°C, generating up to 40 μg of DNA. WGA methods from other suppliers were applied to single-cell samples in parallel. For NGS, 2 μg of DNA was used for shearing and library preparation was done using the TruSeq® DNA Sample Preparation Kit. The libraries were quantified and sequenced (paired-end) on a MiSeq® Instrument. For real-time PCR, 100 pg (bacterial cells) or 10 ng (mammalian cells) of WGA DNA was analyzed using SYBR-Green reagents or RT2 qPCR Arrays. A real-time PCR analysis of 267 loci across the entire genome was performed using 10 ng of WGA DNA for each primer assay. Results showed low and consistent CT-values in real-time PCR for all loci, with no dropout from any marker, indicating successfully amplified DNA from all areas of the genome and high suitability for single-cell genomics. Whole genome sequencing of the Bacillus subtilis genome was performed on the MiSeq from 2 μg of non-amplified genomic DNA or DNA amplified by REPLI-g Single Cell WGA from cells. Comparable sequence coverage was observed for gDNA and REPLI-g Single Cell amplified DNA. A comparison of non-amplified and REPLI-g amplified DNA revealed error rates in a similar, very low, percentage range. The representation of regions of different GC content matches the representation of the genome.