Comparison Of Multiple Displacement Amplification(MDA) And Multiple Annealing And Looping-based Amplification Cycles(MALBAC) In Single-cell Sequencing

Single-cell studies by whole genome amplification were proposed to investigate cellular behaviors from a broad range of environmental and clinical specimens. With the advance of next generation sequencing technologies, single-cell sequencing is expected to result in a novel understanding of genomic stability at the level of per cell cycle in various cell types. There have been several studies on genome-wide sequencing of single cells, which opened an era for investigating haplotype and genomic variability. The quality and quantity of DNA samples are critical for high-throughput sequencing and genetic analysis. Technically, these studies rely on whole-genome amplification from a single cell to generate enough DNA for sequencing library construction. However, the experimental methods employed in previous reports are prone to amplification bias in some degree, resulting in non-random genome coverage. Thus, a crucial innovation in single-cell sequencing is to put in place strategies for whole-genome amplification with minimal amplification bias.Polymerase chain reaction(PCR) based amplification introduces sequencedependent bias due to the exponential amplification with random primers. Multiple displacement w29 DNA polymerase with random primers under isothermal conditions has been widely improved and widely applied in single-cell sequencing projects. Even though, some bias that resulted from non-linear amplification and chimera still exists. Owing to this reason, multiple single-cell sequencings and bulk DNA extractions become necessary for the application of MDA in detecting single nucleotide polymorphisms in diploid genome.An alternative method of whole genome amplification, so-called multiple annealing and looping-based amplification cycles(MALBAC), emerged recently. Combining advantage features of MDA and tweaked PCR, MALBAC substantially reduces experimental bias related to non-linear amplification.To provide a comprehensive comparison of the two methods, we analyzed six human sperm samples from recent single-cell sequencing projects, of which three amplified using MDA and three using MALBAC, respectively. Our results show that MALBAC, as a combination of modified MDA and tweaked PCR, has a higher level of uniformity, specificity and reproducibility.Our study give a scientific and reliable method for monitoring stem cells and tracking of tumor evolution.