Abstract
Single-cell genomic analysis has grown rapidly in recent years and finds widespread applications in various fields of biology, including cancer biology, development, immunology, pre-implantation genetic diagnosis, and neurobiology. To date, the amplification bias, amplification uniformity and reproducibility of the three major single cell whole genome amplification methods (GenomePlex WGA4, MDA and MALBAC) have not been systematically investigated using mammalian cells. In this study, we amplified genomic DNA from individual hippocampal neurons using three single-cell DNA amplification methods, and sequenced them at shallow depth. We then systematically evaluated the GC-bias, reproducibility, and copy number variations among individual neurons. Our results showed that single-cell genome sequencing results obtained from the MALBAC and WGA4 methods are highly reproducible and have a high success rate. The MALBAC displays significant biases towards high GC content. We then attempted to correct the GC bias issue by developing a bioinformatics pipeline, which allows us to call CNVs in single cell sequencing data, and chromosome level and sub-chromosomal level CNVs among individual neurons can be detected. We also proposed a metric to determine the CNV detection limits. Overall, MALBAC and WGA4 have better performance than MDA in detecting CNVs.
Highlights
Sequencing to < 1X coverage is usually performed
Hippocampal neurons were prepared from individual E18 rat embryos and cultured in neurobasal medium as described previously[15,16]
The single neuron nucleus was subjected to whole genome amplification using one of three methods (8 nuclei by multiple annealing and looping-based amplification cycles (MALBAC), 5 nuclei by multiple displacement amplification (MDA) and 6 nuclei by WGA4)
Summary
Sequencing to < 1X coverage is usually performed. Shallow sequencing for identifying CNVs is a common practice reported by several researchers. Previous studies in other groups have reported mixed results of calling chromosome-level CNVs in single cell samples by MDA methods performed in eppendorf tubes. After the quality of single-neuron genome sequencing was confirmed by comparison to the results of traditional sequencing studies (using genomic DNA from ≈ 2 million neurons of the same rat, referred to as bulk cells in this study), we quantitatively analyzed 19 neurons amplified by the WGA4, MDA and MALBAC techniques with an emphasis on the following questions: 1) Is there amplification bias among different genomic regions, and can the bias issue be addressed? Our results demonstrated that single-cell genome sequencing results using either the MALBAC or WGA4 method are highly reproducible and have a high success rate, chromosome-level and sub-chromosomal level CNVs among individual neurons can be detected After the quality of single-neuron genome sequencing was confirmed by comparison to the results of traditional sequencing studies (using genomic DNA from ≈ 2 million neurons of the same rat, referred to as bulk cells in this study), we quantitatively analyzed 19 neurons amplified by the WGA4, MDA and MALBAC techniques with an emphasis on the following questions: 1) Is there amplification bias among different genomic regions, and can the bias issue be addressed? 2) How reproducible are these three whole genome amplification methods? 3) What are the major advantages for each of the three single-cell whole genome amplification methods? Our results demonstrated that single-cell genome sequencing results using either the MALBAC or WGA4 method are highly reproducible and have a high success rate, chromosome-level and sub-chromosomal level CNVs among individual neurons can be detected
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