Decontamination of MDA reagents for single cell whole genome amplification

Abstract

Single cell genomics is a powerful and increasingly popular tool for studying the genetic make-up of uncultured microbes. A key challenge for successful single cell sequencing and analysis is the removal of exogenous DNA from whole genome amplification reagents. We found that UV irradiation of the multiple displacement amplification (MDA) reagents, including the Phi29 polymerase and random hexamer primers, effectively eliminates the amplification of contaminating DNA. The methodology is quick, simple, and highly effective, thus significantly improving whole genome amplification from single cells.

Figure 1. Crossing point (Cp) values for the real-time MDA of single E. coli cells and positive controls using unspiked MDA reagents UV-irradiated for 0, 30, 60 and 90 min.

Figure 2. Shotgun sequence analysis of single E. coli cells amplified with MDA reagents that were UV irradiated for 0, 30, 60 and 90 min (A–C).

Green boxplots represent positive controls and blue boxplots single E. coli cells (A, B). The box is drawn between the first and third quartiles, with the thick black lines representing the median. Dotted lines extend to the minimum and maximum values and outliers are shown as circles. In untreated samples, a large number of sequences mapping to Pseudomonas, Delftia and Stenotrophomonas genomes were found in no template controls (negative controls), as wells a substantial number unmappable reads, which may represent self priming of random hexamers. With 60 min UV irradiation, the contamination in the negative controls was successfully eliminated, leaving no DNA for library generation. Positive controls (10–100 E. coli cells) and individual E. coli cells were free of contamination after 30 min of UV treatment, with ∼98% (median) of reads mapping to the E. coli genome and covering approximately 64% and 21% respectively, which is to be expected at the given sequence effort. (D) Genome coverage rarefaction analysis for the 51 E. coli single cells (UV 0 min, n = 16, UV 30 min, n = 13, UV 60 min, n = 15, UV90 min, n = 7) sequenced shows no significant difference with treatment durations, suggesting that UV irradiation did not negatively impact on the genome recovery. Error bars represent std errors.