Fig 1.
The working principles of ddMDA.
(A) The ddMDA procedures as a high quality alternative to the conventional tube MDA. The MDA ready E. coli samples were partitioned into millions of picoliter droplets using a microfluidic droplet generator. Upon collection, droplets were tightly sealed for isothermal incubation at 30°C for 18 hours. DNA amplicons were then purified, cleaned, and prepared for the following sequencing. (B) Denatured and fragmented whole genomes consist of highly amplifiable (yellow) and weakly amplifiable (red) sequences. During tube MDA, yellow fragments are preferred and repeatedly amplified with a high gain until it reaches a concentration plateau, whereas red fragments are less preferred and barely amplify. For ddMDA, DNA fragments are randomly partitioned into picoliter droplets, resulting in different subsets of the template DNA. When a droplet contains yellow fragments, the amplification kinetics favor the yellow fragments, ending up with significant biases on amplification. The enzyme will amplify red fragments at a slower rate only in the absence of yellow fragments. The overall gain of ddMDA is always lower than tube MDA because of the volume constraint. Every droplet is uniquely composed of fragments and ends up with a different amplification gain after MDA. (C) A fluorescence micrograph showing ddMDA endpoint with the initial template DNA concentration of 100 pg/μL. Having started with different parts of the E. coli genome, individual droplets expressed discrete levels of amplification by showing different sizes of DNA amplicon aggregates and different fluorescent signals. The scale bar shows 100μm.
Fig 2.
Comparison of whole genome coverage of assembled contigs mapped onto E. coli K12 genome sequences for ddMDA and tube MDA.
(A) From the outermost circle, ddMDA for 100 pg/μL (dark purple), tube MDA for 100 pg/μL (bright purple), 10 pg/μL (dark brown), and 10 pg/μL (bright brown), respectively. GC contents across the genome were depicted in black in the innermost circle and reads from genomic DNA were illustrated in green as a reference. (B) From the outermost circle, ddMDA for 1 pg/μL (dark red), tube MDA for 1 pg/μL (bright red), 0.1 pg/μL (dark blue), and 0.1 pg/μL (bright blue), respectively. (C) GC contents (top) and amplification read depths over the entire E. coli genome for ddMDA (middle) and tube MDA (bottom) at the DNA concentration of 10 pg/μL. The right panels show zoomed-in plots of the dotted-line box region of the genome for close-up visualization.
Fig 3.
Improvement of the overall quality of amplification by ddMDA.
(A) The fraction of reads correctly mapped to E. coli genome depending on the DNA concentration and the MDA reaction volume. Blue and red columns show ddMDA and tube MDA results, respectively. The green line shows the fold change of the % mapping from tube MDA to ddMDA (= ddMDA/tubeMDA). It stayed around 1 for high concentrations (10–100 pg/μL) while it considerably increased for low concentrations (0.1–1 pg/μL). (B) The fraction of an E. coli genome covered by one or more sequencing reads depending on the DNA concentration and the MDA reaction volume. Tube MDA and ddMDA showed little difference at high concentrations but the fold change significantly increased at low concentrations. (C) The fraction of an E. coli genome covered by contigs during de novo assembly. While the advantage of ddMDA over tube MDA was still limited at high concentrations but the fold change significantly increased at low concentrations. (D) Lorenz curves depict the amplification bias in read coverage across the E. coli genome. Each curve was calculated by evaluating the read depth for each base and using the resultant cumulative distribution function for read depth to determine the cumulative proportion of total genome coverage (y-axis) accounted for by the cumulative proportion of bases (x-axis). The ideal Lorentz curve (black dotted line) for a distribution in which all of the bases have the same coverage and a Lorenz curve for gDNA were plotted for comparison. Other solid curves show ddMDA curves while dotted curves indicate tube MDA. (Upper Left) For 100 pg/μL, ddMDA in dark purple and tube MDA in bright purple. (Bottom Left) For 10 pg/μL, ddMDA in dark brown and tube MDA in bright brown. (Upper Right) For 1 pg/μL, ddMDA in dark red and tube MDA in bright red. (Bottom Right) For 0.1 pg/μL, ddMDA in dark blue and tube MDA in bright blue.
Table 1.
Statistics of sequence mapping and assembly of E. coli K12 MG1655 samples prepared with ddMDA and conventional tube MDA at different initial copy numbers.
Total number of reads included all sequencing reads mapped and unmapped to E. coli genome. % Reads mapped to genome corresponds to the percentage of sequencing reads that are specifically aligned to E. coli genome. % Genome covered by reads refers to the percentage of E. coli genome covered by one or more sequencing reads. % Genome covered by assembly indicated the percentage of E. coli genome covered by contigs. Gini indices were calculated based on the cumulative distribution of sequencing reads.