Quantitative assessment of single-cell whole genome amplification methods for detecting copy number variation using hippocampal neurons

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.

Scaling by shrinking: empowering single-cell 'omics' with microfluidic devices

“…Other valve-based studies have collectively demonstrated the utility of this approach for performing whole or targeted genome studies in single cells 53–56 . Although current methods are achieving ever more efficient cover age and uniformity, further improvements can still be made to enable truly genome-wide profiling, reduce amplification errors and allow more-confident calling of copy-number variants (CNVs) and single-nucleotide variants (SNVs) 26,57,58 .…”

“…By comparison, proteins, for which consistent and universal probes (typically antibodies) are unavailable, show, on average, higher expression levels than their cognate mRNAs 100 , greater stability 56 and buffering from transcriptional noise 57 . These considerations, coupled with a potentially more direct role in cellular behaviour 27 and the feasibility of non-destructive detection (for secreted and cell-surface-bound factors), have often made protein expression the de facto cellular characteristic.…”

The comparison of the performance of four whole genome amplification kits on ion proton platform in copy number variation detection

“…There are plenty of commercial WGA kits and different kits are based on different strategies: multiple displacement amplification (MDA), degenerate-oligonucleotide-primed PCR (DOP-PCR), and multiple annealing and looping-based amplification cycles (MALBAC, MALBAC-like) are three major WGA strategies. Different strategy may cause different WGA performance and some studies have paid efforts in the comparison of WGA methods [15][16][17][18][19]. However, current related researches are conducted on Illumina sequencing platforms, the results have not validated on Ion Proton platform.…”

Building a lineage from single cells: genetic techniques for cell lineage tracking

“…A second approach, degenerate oligonucleotide priming PCR (DOP-PCR), involves the fragmentation of the genome into small pieces, followed by amplification with random priming 117 . This method amplifies the genome more evenly than MDA, and is thus particularly well-suited to studying copy-number variation 14,85,86,118 . Hybrid methods, including multiple annealing and looping-based amplification cycles (MALBAC) and PicoPlex, include pre-amplification with a tagged primer.…”

“…MALBAC-based amplification is more even across the genome than MDA-based amplification, but error rates are higher 72 . With this more even amplification, hybrid methods are appropriate for investigating CNVs 118 , structural variants 120 and retrotransposition events 79 , although chimeric amplification products that occur in the MALBAC and MDA reactions are of particular concern for interpreting retrotransposition events and structural variants 80 .…”