Consistent temperature-dependent effects on multiple displacement amplification

Multiple displacement amplification (MDA) is widely used in the sample preparation steps of sequencing, but the amplification bias remains a major obstacle to its application. While elevating reaction temperature has been reported to improve MDA performance, the strand thermodynamics across various temperatures remain poorly understood. Through a comparative analysis of the sequencing data generated from MDA reactions performed at different temperatures, we characterized the temperature-dependent patterns of genomic bias and found that performing MDA at 38°C optimizes coverage uniformity for complex human genomic samples. Furthermore, the formation of artificial chimeras under various thermal conditions was quantitatively analyzed. Similar trends were observed in MDA reactions using distinct DNA polymerases, despite the differences in overall amplification efficiency. Together, these results suggest that reaction temperature is a general factor influencing genomic amplification bias during MDA and highlight reaction temperature as an important parameter for improving the uniformity of whole-genome amplification.