Abstract
Whole genome amplification (WGA) is crucial for whole genome sequencing to investigate complex genomic alteration at the single-cell or even single-molecule level. Multiple displacement amplification (MDA) and multiple annealing and looping based amplification cycles (MALBAC) are two most widely applied WGA methods, which have different advantages and disadvantages, dependent on research objectives. Herein, we compared the MDA and MALBAC to provide more information on their performance in droplets and tubes. We observed that the droplet method could dramatically reduce the amplification bias and retain the high accuracy of replication than the conventional tube method. Furthermore, the droplet method exhibited higher efficiency and sensitivity for both homozygous and heterozygous single nucleotide variants (SNVs) at the low sequencing depth. In addition, we also found that MALBAC offered a greater uniformity and reproducibility and MDA showed a better efficiency of genomic coverage and SNV detection. Our results provided insights that will allow future decision making.
Highlights
High throughput sequencing has tremendously influenced biomedical research due to its ability to acquire massive amounts of sequence data [1,2,3]
We found that multiple annealing and looping based amplification cycles (MALBAC) significantly improved the uniformity and reproducibility while Multiple displacement amplification (MDA) shown a better efficiency in genomic coverage and single nucleotide variants (SNVs) detection
To validate that the droplet system enabled massively parallel to generate reaction environments within limited DNA samples, MDA and MALBAC mixtures were emulsified with 12.500 ng DNA and without DNA, respectively
Summary
High throughput sequencing has tremendously influenced biomedical research due to its ability to acquire massive amounts of sequence data [1,2,3]. MALBAC uses random priming and poikilothermic preamplification in the early stage, followed by PCR amplification with limited circles [19,20] It suppresses the random bias of amplification and exhibits reduced allelic dropout rate [20,21], coverage and uniformity are both improved by virtue of quasilinear amplification intermediately [22]. These approaches can be performed combining with the microfluidic method, which has extensively been applied to genomics [23,24], proteomics [25,26], metabonomics [27,28]. Our results can provide a guidance to choose a better method
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