Fig. 2: Simulation comparison between MPHAC and conventional fixed length primer amplification.
a Employing MPHAC for selective amplification of microchip-synthesized DNA to enhance molecular quantity while maintaining sequence homogeneity. b Relationship between primer amplification efficiency and hybridization yield. c In the presence of two files of equal size, the variation in the file ratio before and after amplification (defined as ratio change) spans over 10 orders of magnitude, influenced by different PCR cycles and their respective amplification efficiencies. d Ratio change at various amplification efficiencies with 20 PCR cycles. Conventional pathway is often based on fixed-length (FL) primers, while MPHAC achieves consistent amplification efficiency by rigorously controlling the primers with fixed-energy (FE). For comparing conventional pathway with MPHAC, we randomly generated 100,000 primer pairs using both methods based on the human genome sequence, and simulated their performance. e Thermodynamic energy distribution of FL-primers and FE-primers. Analysis of primer hybridization thermodynamic energy indicates that, even with a fixed length and restricted GC content, the free energy distribution of the primers remains wide, causing variations in their amplification efficiencies. f Simulated sequencing results of FL-primers under 50× sequencing depth. Using a 30× threshold, detecting all sequences requires an 11,643-fold deeper sequencing depth. g Simulated sequencing results of FE-primers under 50× sequencing depth. In contrast, MPHAC achieves consistent amplification efficiency by rigorously controlling the primer free energy distribution, ensuring the effective detection of all sequences. h Comparison of ratio consistency before and after amplification using FL-primers vs. FE-primers. r2 is the square of Pearson’s correlation coefficient, calculated from the total sum of squares and the residual sum of squares. i Performance comparison between FL-primers (blue) and FE-primers (red). The calculation for each metric is described in the “Methods section”. Source data are provided as a Source Data file.
