Ecological analysis from DGGE profiles generated

Each commercially available DNA polymerase enzyme was assessed in all five sandy soil samples by employment of technical replicates to ensure PCR bias was countered for (within a certain degree) when the appropriate statistical analysis was performed (below). Ecological analysis of the bacterial community generated in each DGGE profile varied. Isolated distinct bands migrating through the denaturing gradient were treated as a single bacterial taxon from which the mean species richness and diversity indices (i.e., Shannon diversity index scores (H′)) was calculated from the technical replicates of the five different DNA pol enzymes in addition to the average number of bands detected (summarised in table 3.2).

Across sample numbers 1-5, both high-fidelity enzymes Ex Taq™ HS and Pfx generated the greatest mean number of bands in each DGGE profile analysed and highest mean H′ scores (the only exception being in SS3R where the mean H′ score (2.30) of Ex Taq™ HS was equivalent to GoTaq® HS). Applied Biosystems™ AmpliTaq® detected the lowest number of bands (only 1 in SS5R as opposed to Platinum® Pfx in which 20.7 bands were detected on average) across all five samples in addition to the lowest mean H′ scores, again, taking into consideration that no H′ indices could be calculated for SS5R (each AmpliTaq® replicate only detected one bacterial taxa) and in SS4R as previously mentioned above. Overall bacterial diversity in the five sandy soil sample types is summarised in table 3.3. SS3R yielded the highest detection of bacterial diversity (H′ range 0.66-3.16 (mean = 2.31)) in addition to also showing the highest band range (2-25 (mean = 13.5)) whereas SS4R displayed the lowest (H′ range 0.61-3.03 (mean = 1.95)). Both SS3R and SS4R produced a wide range in their H′ scores, it should be noted however that in two of the AmpliTaq® technical replicates SS4R and SS5R (i.e., all AmpliTaq® replicates) Shannon diversity indices could not be derived as only one bacterial taxa each was detected.

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Table 3.2: Comparison of mean number of bands detected and H′ scores calculated using technical replicates of the five DNA polymerase enzymes assessed.

Sample replicate

number

DNA pol enzyme§

Mean number of bands detected by each DNA

pol

Mean H′ score generated by each DNA pol (n = 5)

SS1R Taq 7.7 1.94 GoTaq® HS 12 2.29 AmpliTaq® 4 1.29 Ex Taq™ HS 18.3 2.83 Pfx 16 2.65 SS2R Taq 7 1.84 GoTaq® HS 10.7 2.30 AmpliTaq® 3.7 1.27 Ex Taq™ HS 11.7 2.30 Pfx 12 2.42 SS3R Taq 9.3 2.16 GoTaq® HS 11.7 2.37 AmpliTaq® 3.7 1.06 Ex Taq™ HS 18 2.80 Pfx 25 3.16 SS4R Taq 8 1.94 GoTaq® HS 10 2.20 AmpliTaq® 1.3 0.20 Ex Taq™ HS 21 3.02 Pfx 14 2.39 SS5R Taq 11.3 2.36 GoTaq® HS 9.7 2.16 AmpliTaq® 1 0† Ex Taq™ HS 17.7 2.76 Pfx 20.7 2.87

§ In order of loading on DGGE profile generated.

† Average H′ score cannot be derived if only one bacterial taxon is detected across all three AmpliTaq® technical replicates.

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Table 3.3: Comparison of bacterial diversity in the five sandy soil samples analysed by DGGE. Sample replicate number Band range per lane Mean number of bands per lane

H′ range across sample Mean H′ across sample SS1R 4-19 11.6 1.23-2.88 2.20 SS2R 4-13 9 1.10-2.52 2.03 SS3R 2-25 13.5 0.66-3.16 2.31 SS4R 1-21 10.9 0.61-3.03 1.95 SS5R 1-21 12.1 2.01-2.95 2.03

Qualitatively, each DGGE profile generated (i.e., SS1R to SS5R) yielded similar banding patterns from bacterial taxa detected by the five individual DNA pols assessed, including the technical replicates (Fig. 3.2A-E). Applied Biosytems™ AmpliTaq® performance was very poor in comparison to the other DNA pol assessed due to the distinct lack of bands throughout the denaturing gradient, in particular, the bands that were detected are faint (white arrows, Fig. 3.2C). New England BioLabs® Inc. Taq and Promega’s GoTaq® HS both produced approximate equivalent banding patterns from their technical replicates in all DGGE profiles (Fig. 3.2A-E). Both high-fidelity DNA pol enzymes Ex Taq™ HS and Platinum® Pfx detected the most abundant bacterial taxa from the dominant members of the community as seen in all gels below resulting in a greater mean band range across all five sample types. Amplification of the V3 rDNA regions performed with Pfx did result in more bacterial taxa being detected in SS2R, SS3R, and SS5R. Interestingly, bands were notably absent in the upper 25 % of the denaturing gradient (i.e., near the top of the gel in Fig. 3.2A- E, circled) in almost all Pfx technical replicates and thus consequently not detected. TAKARAS BIO INC. Ex Taq™ HS in SS1R-SS5R in this regard produced a more widespread detection of bacterial taxa throughout the 35-55 % denaturing gradient utilised (and indeed more numerous specifically in SS1R and SS4R), in addition to taxa that migrated towards the top of the gradient. A global factor in all of the DNA pol enzymes assessed is that band detection in the lower part of the denaturing gradient seemed to be more abundant in the derived Thermus aquaticus DNA pol types despite the band number, whereas in Pfx, derived from Pyrococcus spp. strain KOD1, detected a greater number of bands in the middle

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of the gradient. All DGGE profiles generated showed heteroduplex artefacts (i.e., double band formations that migrate through the denaturing gradient very close together) and these have been putatively identified with examples shown in Fig. 3.2A, B, and D. These double bands were common in all Taq, GoTaq® HS, and Ex Taq™ HS technical replicates across all five sample types.

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A

B

Taq GT AB ET Pfx

Fig. 3.2A-E: DGGE profiles (35-55 % denaturing gradient) of SS1R-SS5R showing V3 rDNA fragments from all DNA pol technical replicates assessed. SS1R = A; SS2R = B; SS3R = C; SS4R = D, and SS5R = E. Top of the denaturing gradient is shown (35 %) along with the bottom (55 %). Migration direction of the amplicons and denaturing gradient is indicated on the left in gels A-E (arrow). Technical replicates of each DNA pol enzyme are indicated above each DGGE profile (A-E) respectively. Non-detection of bands by Pfx in all technical replicates across SS1R-SS5R is circled. Examples of putative artefacts in DGGE profiles are shown (black arrows) whereas detection of taxa by AmpliTaq® is indicated because of faint bands produced in gel profiles (white arrows). AB, AmpliTaq®; ET, Ex Taq™ HS; GT, GoTaq® HS.

55% 35%

55% 35%

73 35%

_C

D

74 35%

55%

E

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