Comparison of six commercial kits to extract bacterial chromosome and plasmid DNA for MiSeq sequencing

Abstract

We compared commercial kits for extraction of genomic DNA from the Gram-negative bacterium Klebsiella pneumoniae for subsequent Miseq sequencing. Purification of DNA was based on matrix binding (silica or anion exchange resin) or differential precipitation (salting out), respectively. The choice of extraction kit had little effect on sequencing quality and coverage across drastically different replicons, except for an apparent depletion of small plasmids (<5 kb) during precipitation-based extractions. Sequencing coverage provided copy-number estimates for small plasmids that were consistently higher than those from quantitative real-time PCR.

Figure 1. Pulsed-field gel electrophoresis of K. pneumoniae 234-12 DNA extracted with six different kits.

DNA was extracted from aliquots of the same overnight culture according to the manufacturers′ protocols, and 10 μl of resulting extracts were loaded per lane. M1 Size standard Salmonella Braenderup lysed in agarose plug, DNA digested with XbaI. 1 K. pneumoniae 234-12 lysed in agarose plug, DNA digested with S1 nuclease for presentation of the linearized 362-kb plasmid. 2 Genomic-tip 20/G. 3 MagAttract HMW DNA Kit. 4 MasterPure DNA Purification Kit. 5 Wizard Genomic DNA Purification Kit. 6 DNeasy Blood & Tissue Kit. 7 Plasmid Mini Kit. M2 GeneRuler 1 kb Plus DNA Ladder (Thermo Scientific).

Figure 2. Effect of DNA extraction kit on the sequencing coverage of the chromosome and the three plasmids of K. pneumoniae 234–12.

Means and standard deviations from three independent experiments are reported. For statistical analysis, two-tailed student’s t-tests with Bonferroni correction were performed (global significance level α = 0.10). Asterisks indicate a statistically significant difference compared to the DNeasy kit (p-value below 0.02). One gene was excluded from this evaluation, because orthologues were found on both the chromosome and the 3.8-kb plasmid.

Figure 3. Coverage along replicons.

Bars correspond to the mean coverage of 10,000 nucleotides for the chromosome, 1,000 nucleotides for the 362-kb plasmid and 100 nucleotides for the two small plasmids, respectively. Pointed regions (red arrows) contain a gene occurring on both the 3.8-kb plasmid and chromosome.