Optimization of computer software settings improves accuracy of pulsed-field gel electrophoresis macrorestriction fragment pattern analysis

Abstract

Computer-assisted analysis of pulsed-field gel electrophoresis (PFGE) libraries can facilitate comparisons of fragment patterns present on multiple gels. We evaluated the ability of the Advanced Analysis (version 4.01) and Database (version 1.12) modules of the Phoretix gel analysis software package (Nonlinear USA, Inc., Durham, N.C.) to accurately match DNA fragment patterns. Two gels containing 38 lanes of SmaI-digested Enterococcus faecalis OG1RF DNA were analyzed to assess the impact of (i) varying the lane position of the standards, (ii) using gel plugs made at different times, and (iii) normalizing the fragment patterns by using molecular weight (MW) algorithms versus retardation factor (R(f)) algorithms. Two sets of PFGE libraries (one containing SmaI restriction patterns from 62 Enterococcus faecium isolates and the other containing SmaI restriction patterns of 89 Staphylococcus aureus isolates) were analyzed to assess the impact of varying the matching tolerance algorithm (designated as the vector box setting [VBS]) in the Phoretix software. Varying the lane position of standards on a gel and using gel plugs made on different days resulted in different VBSs, although it was not possible to judge whether those differences were statistically significant. Normalization of E. faecalis OG1RF fragment patterns by R(f) and MW methodology yielded no statistically significant differences in variability between the same fragment on different lanes. Suboptimal VBSs decreased the specificity with which related isolates were grouped together in dendrograms. The optimal VBS for analysis of PFGE fragment patterns from E. faecalis isolates differed from that for S. aureus isolates and sometimes was not that recommended by the manufacturer. Thus, computer-assisted analysis of PFGE patterns seemed to compensate for the intra- and intergel variation evaluated in the present study, and optimizing the software for the species to be tested was a critical preliminary step before further PFGE library analysis.

FIG. 1.

Gel 2: lanes containing SmaI-digested E. faecalis OG1RF DNA. Some lane-to-lane variations, or distortions, exist in gel 2. Distortions are pronounced in lanes 4 to 6.

FIG. 2.

Comparison of fragment-to-fragment matching at two vector box sizes of 0.500 and 0.925%. At a VBS of 0.500%, fragment 5 of lane X will match fragment 5 of lane Y, but fragment 7 of lane X does not match fragment 6 of lane Y. At a VBS of 0.925%, fragment 7 of lane X does match fragment 6 of lane Y due to fragment overlap caused by increasing VBS.

FIG. 3.

UPGMA dendrogram generated from Dice coefficients that contains E. faecium PFGE library isolates compared at a VBS of 0.925%. Isolates marked with an asterisk indicate those in the VSG. The single isolate indicated by a “^” symbol (designated 1435 in the middle of the dendrogram group) is the reference (index) isolate. Of 31 isolates in the dendrogram group (marked with a bracket), 26 were visually similar, and 5 were dissimilar. The match similarity for the dendrogram group was 74%.

FIG. 4.

UPGMA dendrogram generated from Dice coefficients that contains S. aureus PFGE library isolates compared at a VBS of 0.500%. Isolates marked with an asterisk indicate those in the visually similar group (VSG). The single isolate indicated by a “^” symbol (designated lane 19 toward the top of the dendrogram group) is the reference (index) isolate. Of 35 isolates in the dendrogram group (marked with a bracket), 33 were visually similar, and 2 were dissimilar. The match similarity for the dendrogram group was 79%.