The PFGE patterns were analyzed using FPQuest software version 4.5 (Bio-Rad) by methods described earlier [38] (link). Similarity analysis was done by using Dice coefficient with 1.5% optimization and tolerance levels and clustering of matched bands was done using Unweighted Pair Group Method with Arithmetic mean (UPGMA). The isolates with identical PFGE patterns were described as genetically indistinguishable (Dice coefficient of similarity of 100%); isolates with PFGE patterns differing by three or less bands were designated as related (Dice coefficient of similarity of >80%).
Fpquest software version 4
Manufactured by Bio-Rad
Sourced in United States, United Kingdom
FPQuest software version 4.5 is a data analysis tool for fluorescence polarization (FP) assays. It provides functions to acquire, analyze, and visualize FP data generated from experiments.
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Lab products found in correlation
5 protocols using fpquest software version 4
1
Pulsed-field Gel Electrophoresis Analysis
2
Pulsed-Field Gel Electrophoresis for NDM-1 Isolates
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PFGE was carried out for all NDM-1-possessing isolates by following PulseNet standardized procedures (http://www.cdc.gov/pulsenet/protocols.htm ) in a CHEF-DR III apparatus (Bio-Rad Laboratories, Hercules and CA). XbaI macrorestriction patterns were compared and interpreted according to the criteria of Tenover et al.[26] . The dendrogram was generated by FPQuest software, version 4.5 (BioRad Laboratories, Hercules, CA, USA).
3
Bacterial Genomic DNA Fingerprinting
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PFGE of XbaI digested genomic DNA of the study isolates was performed, using CHEF DRIII (Bio-Rad), following the PulseNet standard protocol [43 ]. S. Braenderup H9812 was used as reference strain. The PFGE profiles were analyzed using FP Quest software version 4.5 (Bio-Rad). The extent of homology was determined by Dice coefficient with 1.5% optimization and tolerance levels, and clustering of bands was done by Unweighted Pair Group Method with Arithmetic mean (UPGMA). Based on PFGE profiles, one PFGE pulsotype (PP) number was assigned to each isolate. The isolates with identical PFGE patterns (Dice coefficient of similarity of 100%) were described as genetically indistinguishable; isolates with PFGE patterns differing by three or less bands (Dice coefficient of similarity of >80%) were designated as related and grouped under one cluster [44 (link)].
4
Molecular Characterization of S. aureus Strains
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From the 260 representative strains, DNA extraction was performed into agarose disks and strains were classified into pulsotypes by pulsed-field gel electrophoresis (PFGE) of chromosomal DNA SmaI digests performed in a CHEF DR III apparatus (Bio-Rad, Richmond, CA), as previously described [41 (link)]. A dendrogram comparing molecular weights of strains’ DNA fragments was performed by FPQuest software version 4.5 (Bio-Rad Laboratories Inc). According to criteria established by Miragaia et al. [42 (link)] patterns differing by less than 79 % (corresponding to a difference of less than seven bands) were considered to belong to the same PFGE type.
The selected S. aureus strains were also characterized by Multilocus Sequence Typing (MLST) (http://mlst.net ) [43 (link)]. The results were analyzed by the application of eBURST algorithm. Clonal complexes were defined by using the default setting, in which all STs within a clonal complex differ by no more than one allele from at least one other ST in the clonal complex.
The selected S. aureus strains were also characterized by Multilocus Sequence Typing (MLST) (
5
Microbial Community Analysis of W-DDGS Diets
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Data were subjected to analysis of variance (ANOVA) using a fully randomised design Genstat v14 (VSN, International Ltd, Hemel Hempstead, UK) with diet as the main factor, with linear and non-linear contrasts to account for the incremental increase in W-DDGS.
Unweighted Pair Group Method with Arithmetic Mean (UPGMA) dendrograms of DGGE banding patterns were generated by FPQuest Software Version 4.5 (BIO-RAD Laboratories) using the Dice coefficient. Analysis of molecular variance (AMOVA) was performed to compare the DGGE patterns of bacteria communities at a selected similarity level as according to Excoffier et al. (1992) using GenAIEX v6.5 software as described by Peakall and Smouse (2012) . The Shannon-Wiener diversity index (H) was used to describe bacterial diversity as detected by DGGE (Scanlan et al., 2006 , Shannon, 1948) .
Unweighted Pair Group Method with Arithmetic Mean (UPGMA) dendrograms of DGGE banding patterns were generated by FPQuest Software Version 4.5 (BIO-RAD Laboratories) using the Dice coefficient. Analysis of molecular variance (AMOVA) was performed to compare the DGGE patterns of bacteria communities at a selected similarity level as according to Excoffier et al. (1992) using GenAIEX v6.5 software as described by Peakall and Smouse (2012) . The Shannon-Wiener diversity index (H) was used to describe bacterial diversity as detected by DGGE (Scanlan et al., 2006 , Shannon, 1948) .
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