A primer-independent DNA polymerase-based method for competent whole-(meta)genome amplification of intermediate to high GC sequences

Abstract

Multiple displacement amplification (MDA) has proven to be a useful technique for obtaining large amounts of DNA from tiny samples in genomics and metagenomics. However, MDA has limitations, such as amplification artifacts and biases that can interfere with subsequent quantitative analysis. To overcome these challenges, alternative methods and engineered DNA polymerase variants have been developed. Here, we present new MDA protocols based on the primer-independent DNA polymerase (piPolB), a replicative-like DNA polymerase endowed with DNA priming and proofreading capacities. These new methods were tested on a genomes mixture containing diverse sequences with high-GC content, followed by deep sequencing. Protocols relying on piPolB as a single enzyme cannot achieve competent amplification due to its limited processivity and the presence of ab initio DNA synthesis. However, an alternative method called piMDA, which combines piPolB with {Phi}29 DNA polymerases, allows proficient and faithful amplification of the genomes. In addition, the prior denaturation step commonly performed in MDA protocols is dispensable, resulting in a more straightforward protocol. In summary, piMDA outperforms commercial methods in the amplification of metagenomes containing high GC sequences and exhibits similar profiling, error rate, and variant determination as the non-amplified samples. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/533076v2_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1cfb02dorg.highwire.dtl.DTLVardef@378d8corg.highwire.dtl.DTLVardef@12e0f93org.highwire.dtl.DTLVardef@1008ffd_HPS_FORMAT_FIGEXP M_FIG C_FIG Schematic representation of methods based on multiple displacement amplification (MDA) for whole genome amplification. The diagrams above represent protocols initiated by random primers (RP-MDA) or a DNA primase-generated short DNA primers (PrimPol-MDA) and continued by {Phi}29DNAP, whereas the schematics below show piPolB-mediated MDA (left) and the piMDA protocol (right), in which piPolB synthesizes DNA strands that are further extended by {Phi}29DNAP.