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Multiple displacement amplification (MDA) is considered to be a conventional approach to comprehensive amplification from low input DNA. The chimeric reads generated in MDA lead to severe disruption in some studies, including those focusing on heterogeneity, structural variation, and genetic recombination. Meanwhile, the generation of by-products gives a new approach to gain insights into the reaction process of φ29 polymerase. Here, we analyzed 36.7 million chimeras and screened 196 billion chimeric hotspots in the human genome, as well as evaluating the hotspot selective preference of chimeras. No significant preference was captured in the distributions of chimeras and hotspots among chromosomes. Hotspots with overlaps for 12-13 nucleotides (nt) were most likely to be selected as templates in chimera generation. Meanwhile, a regularly selective preference was noticed in overlap GC content. The preferences in overlap length and GC content was shown to be pertinent to the sequence denaturation temperature, which pointed out the optimization direction for reducing chimeras. Distance preference between two segments of chimeras was 80-280 nt. The analysis is beneficial for reducing the chimeras in MDA, and the characterization of MDA chimeras is helpful in distinguishing MDA chimeras from chimeric sequences caused by disease.

Hotspot Selective Preference of the Chimeric Sequences Formed in Multiple Displacement Amplification