Negative effects of sample pooling on PCR-based estimates of soil microbial richness and community structure

Abstract

In this study, we examined the effect of various pooling strategies on the characterization of soil microbial community composition and phylotype richness estimates. Automated ribosomal intergenic spacer analysis (ARISA) profiles were determined from soil samples that were (i) unpooled (extracted and amplified individually), (ii) pooled prior to PCR amplification, or (iii) pooled prior to DNA extraction. Regression analyses suggest that the less even the soil microbial community (i.e., low Shannon equitability, E(H)), the greater was the impact of either pooling strategy on microbial detection (R(2) = 0.766). For example, at a tropical rainforest site, which had the most uneven fungal (E(H) of 0.597) and bacterial communities (E(H) of 0.822), the unpooled procedure detected an additional 67 fungal and 115 bacterial phylotypes relative to either of the pooled procedures. Phylotype rarity, resulting in missed detection upon pooling, differed between the fungal and bacterial communities. Fungi were typified by locally abundant but spatially rare phylotypes, and the bacteria were typified by locally rare but spatially ubiquitous phylotypes. As a result, pooling differentially influenced plot comparisons, leading to an increase in similarity for the bacterial community and a decrease in the fungal community. In conclusion, although pooling reduces sample numbers and variability, it could mask a significant portion of the detectable microbial community, particularly for fungi due to their higher spatial heterogeneity.

FIG. 1.

Site-averaged rarefaction curves for fungi (A to C) and bacteria (D to F) determined using three sampling strategies. Filled circles, no pooling; open circles, pooled prior to PCR; filled triangles, pooled prior to DNA extraction. At each site, three separate rarefaction curves were generated using the nine biological (no pooling) or nine technical (pooled procedures) replicates from each plot. Sites represented in the panels are as follows: ARDEC corn field (A and D), TNR tropical rainforest (B and E), and YG ponderosa pine forest (C and F).

FIG. 2.

Average Jaccard similarity index (J_class) between the three plots at each site (ARDEC corn field, TNR tropical rainforest, and YG ponderosa pine forest). J_class (phylotype presence/absence) was calculated between the three plots at each site using each individual ARISA profile (A and C) or a single composite ARISA profile (i.e., average of all nine biological [no pooling] or nine technical [pooled procedures] replicates) for each plot (B and D). Averages are reported by site (A and B) or sampling procedure (C and D). Black bars, fungi; gray bars, bacteria. Bars are the LSmeans with the pooled standard error (SE); bars with different letters denote significant Tukey's HSD test results (P < 0.05).