Here, we present two broadly applicable methods for direct detection of microbial communities in natural environments, both of which provide cost-effective alternatives to PCR-amplified microbial community analysis. Based on tests with samples from groundwater, soil, and secondary sewage in seawater, these methods should be applicable to many diverse types of environmental or clinical samples, with good resolution of the different sample types that do not parse by method (). Starting with a genetic pool that is free from bias and distortion introduced from PCR amplification and combining this with the rapid, reproducible, and cost-effective microbial community analysis of high-density microarrays create a powerful method for efficiently querying myriad microbial communities with replication, as well as analysis tuned for maximum statistical power and fidelity of microbial community dynamics.
Fig. 4. Ordination of dscDNA- and PCR-DNA-derived microbial communities from chromium-contaminated groundwater, anaerobic incubations of tropical forest soils, and secondary sewage sludge incubated in seawater. Each point is one biological replicate; filled symbols (more ...)
The mock-community analysis contained evidence of cross-hybridization, an issue which has been previously observed (18
), and in both studies, false positives were largely limited to the family level. While this could pose problems for low-richness samples, overinflated richness of closely related sequences is a problem for many kinds of community analysis (9
), and increased sampling tends to result in increased apparent richness based on nonasymptotic accumulation curves (6
). Though more diversity should not be captured in a community of limited richness, contamination is possible in strains without selective markers and was detected with pyrosequencing; even culture collections of isolates can carry trace impurities that would be detectable by this sensitive method. Both a denoising algorithm and a new version of PhyloChip analysis that eliminates cross-hybridization seem to reduce this problem (27
We observed decreased taxon richness detected by the RNA-based methods compared to the PCR-DNA method, with the exception of the dscDNA sewage samples, which suggests that only a subset of the standing community is active. That the T0 sewage samples had equal levels of richness in active (by dscDNA) and standing (by PCR-DNA) communities suggests that the active community is as diverse as the standing reservoir community. We observed fidelity in community profiles within an environment across different methods of microbial community detection, evidenced by ordination or hierarchical clustering that separated dscDNA and PCR-DNA communities. Though the dirRNA and PCR-DNA communities grouped by replicate while dscDNA communities grouped by time point (), we detected more taxa with decreasing metabolic activity over time in the dirRNA and dscDNA communities than in the PCR-DNA communities (see Table S6 in the supplemental material). This could be because the RNA-based methods are more sensitive in detecting changes in active microbial populations. These observations are consistent with the properties of the sewage community, where the inactive bacterial DNA could persist in the environment for prolonged periods of time (29
). Likewise in the unamended tropical soil-derived microcosms, there were more phyla with significantly lower richness in the RNA communities than in the PCR-DNA communities (see Table S4). There are some instances where the RNA methods detected organisms not detected by PCR (see, e.g., Table S7), which could represent highly active organisms, or relatively low concentration organisms, or both. The PCR-DNA method is thus more a measure of composition, where the bias of amplification magnifies the contribution of rare species, whereas the dscDNA data reflect the metabolically active populations.
Examination of microbial communities based on both rRNA and DNA (rRNA genes) revealed different members in each pool, with some taxa detected by one method but not the other. Taxa detected by the RNA methods but not the PCR-DNA method could be rare species that are highly active, that contain high concentrations of rRNA, or that contain sequences not amplifiable by our primers. Taxa detected only by PCR (DNA only) are likely largely inactive but abundant (38
). While rRNA gene copies are generally constrained within a given species, rRNA abundance can vary by up to 5 orders of magnitude, depending upon the activity of the population (32
), and may change rapidly, even in a few hours (39
). Variations in 16S rRNA gene copy numbers between species are at least partially responsible for differences in growth rates (32
), as could be polyploidy in rapidly multiplying cells, but because the rRNA concentrations change over shorter time scales due to changes in activity, the RNA/DNA ratio (or dscDNA/DNA, as we used here) may be used as an indicator of cellular activity for single species (40
) and has been applied to microarray data (16
). The few known exceptions to the linear relationship between activity and rRNA abundance seem to be at least partially related to unusual growth conditions not typical of a natural environment (3
) though all measures of this relationship so far have been in culture. The microarray method has a low detection limit of 107
copies, or about 0.01% of the total nucleotides applied (7
), so the lack of detection of certain taxa in one nucleic acid pool over the other is interpreted to mean that the rRNA dipped below the detection limit rather than being actually absent, allowing us to infer activity status for an even wider swath of the microbial community.
The two methods presented, direct hybridization of RNA (dirRNA) and direct hybridization of double-stranded cDNA (dscDNA), are both valid ways of exploring microbial community structures, each with advantages and disadvantages. RNA direct hybridization involves the least manipulation of environmental nucleic acids, but the short half-life that makes RNA useful for encoding messages in cells also makes it difficult to work with. This was a limiting factor in the tropical forest soil lab incubations, where low activity resulted in low RNA yields and precluded some analyses (A). The dscDNA method employs essentially one round of amplification, which should remain free of the amplification biases that impact analysis of communities by PCR amplification, which typically requires 25 to 35 amplification rounds before analysis; primer binding efficiency may play a role though we assume that it is the same per sequence across different environments. The dscDNA method has an additional advantage in that the chemistry in the labeling and hybridization reactions is the same as for the PCR-amplified DNA reaction, which has been employed successfully since 1996 (10
), has been in use with the PhyloChip since 2003, and now claims 41 publications at the time of this writing (6
). The different labeling reagent and hybridization conditions required for RNA direct hybridization result in a slightly lower quality scan (see Fig. S1 in the supplemental material) and, while still within the limits of quality control, likely results in lower reproducibility and higher variability between samples.
There is some precedent for direct hybridization of rRNA onto microarrays, which have been in development for detection of rRNA and rRNA genes derived from complex microbial communities for over 15 years (25
). Proof-of-principle for reproducible and specific detection of microbial communities from the environment was established by Small et al. with pure cultures of Geobacter chapellei
and Desulfovibrio desulfuricans
by performing direct rRNA hybridization onto a custom microarray fabricated for the detection of these two metal-reducing strains with bioremediative potential (49
). The work of Small et al. supported the specificity obtainable with direct hybridization but ran into problems with reproducibility, variability, and background. Our method of dscDNA direct hybridization offers a solution to this problem with minimal additional sample manipulation and at a lower cost than PCR amplification (see Fig. S1 and Table S7 in the supplemental material). Direct hybridization of large-subunit (LSU) rRNA developed for microbial community analysis by microarray has also been recently presented, and the authors experienced problems with background and nonspecific hybridization (41
). The dscDNA direct hybridization method presented is a novel solution to the minor issues involved with direct rRNA hybridization, with both methods conforming to established quality control measures. Ultimately, it is left to the investigators to choose the method of direct hybridization based on the unique qualities of their own experimental design and samples, knowing that PCR amplification-free microbial community analysis provides a faithful and cost-effective representation of naturally occurring, metabolically active microbial communities.
Direct hybridization of rRNA (dirRNA) and rRNA-derived double-stranded cDNA (dscDNA) to high-density microarrays represents a simple and economical way to directly query microbial communities in natural environments. Microbial communities revealed by direct hybridization display different profiles from the corresponding PCR-amplified DNA communities, likely reflecting both PCR amplification biases and differences in the microbiology of active versus inactive populations in environmental samples. Both methods offer viable alternatives to PCR-amplified microbial community profile methods and offer the additional advantage of detecting activity in populations rather than the simple presence of organisms. Specifically targeting active members of microbial communities facilitates identification of microbial taxa that attenuate toxins in the environment, drive biogeochemical cycles in natural systems, or proliferate in disease states.