Dataset Information

Two-stage microbial community experimental design.

ABSTRACT: Microbial community samples can be efficiently surveyed in high throughput by sequencing markers such as the 16S ribosomal RNA gene. Often, a collection of samples is then selected for subsequent metagenomic, metabolomic or other follow-up. Two-stage study design has long been used in ecology but has not yet been studied in-depth for high-throughput microbial community investigations. To avoid ad hoc sample selection, we developed and validated several purposive sample selection methods for two-stage studies (that is, biological criteria) targeting differing types of microbial communities. These methods select follow-up samples from large community surveys, with criteria including samples typical of the initially surveyed population, targeting specific microbial clades or rare species, maximizing diversity, representing extreme or deviant communities, or identifying communities distinct or discriminating among environment or host phenotypes. The accuracies of each sampling technique and their influences on the characteristics of the resulting selected microbial community were evaluated using both simulated and experimental data. Specifically, all criteria were able to identify samples whose properties were accurately retained in 318 paired 16S amplicon and whole-community metagenomic (follow-up) samples from the Human Microbiome Project. Some selection criteria resulted in follow-up samples that were strongly non-representative of the original survey population; diversity maximization particularly undersampled community configurations. Only selection of intentionally representative samples minimized differences in the selected sample set from the original microbial survey. An implementation is provided as the microPITA (Microbiomes: Picking Interesting Taxa for Analysis) software for two-stage study design of microbial communities.

SUBMITTER: Tickle TL

PROVIDER: S-EPMC3834858 | biostudies-other | 2013 Dec

REPOSITORIES: biostudies-other

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Two-stage microbial community experimental design.

Tickle Timothy L TL Segata Nicola N Waldron Levi L Weingart Uri U Huttenhower Curtis C

The ISME journal 20130815 12

Microbial community samples can be efficiently surveyed in high throughput by sequencing markers such as the 16S ribosomal RNA gene. Often, a collection of samples is then selected for subsequent metagenomic, metabolomic or other follow-up. Two-stage study design has long been used in ecology but has not yet been studied in-depth for high-throughput microbial community investigations. To avoid ad hoc sample selection, we developed and validated several purposive sample selection methods for two- ...[more]

PMID: 23949665

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Project description:Two consortia (Consortium A and Consortium B) that can use 1,4-dioxane (a groundwater contaminant of emerging concern) as the sole carbon source were enriched from Rice University (Houston, TX, USA) campus soil. Phylogenetic analysis by 16S rRNA sequencing revealed the dominant genus in both of the consortia is Mycobacterium (56% in Consortium A and 49% in Consortium B). The predominance of Mycobacterium spp, in these consortia support the notion that this is an important and commonly encountered genus of dioxane degraders. Among other genera present that make at least 2% of these consortia, only Afipia encompasses a strain (i.e., Afipia sp. D1) that was reported to degrade dioxane as sole carbon and energy source. A nested PCR analysis using two degenerate primers to target the hydroxylase alpha subunit of groups 3 to 6 SDIMOs was performed to gain insights into which enzymes were responsible for dioxane degradation by these consortia. The purified products obtained from the second PCR run were sequenced and compared to genes databases (NCBI) encompassing all of the currently reported SDIMOs. The dominant SDIMO genes in Consortium A corresponded to a group-6 putative propane monooxygenase-like SDIMO (98.8%); while in Consortium B, SDIMO genes from both groups 5 (47.3%) and 6 (51.9%) were observed. In both consortia, the relative abundance of thmA/dxmA gene was negligible (0.03%), which is consistent with the negative amplification of these genes as verified in qPCR. Overall, the high relative abundance of group-6 putative propane monooxygenases in our two consortia suggests the novel finding that group 6-SDIMOs could also play an important role in dioxane degradation. This underscores the need for further research on genes and enzymes involved in dioxane biodegradation to develop novel biomarkers that can be useful for forensic analysis and performance assessment of bioremediation and natural attenuation at dioxane-impacted sites. DNA was extracted from bacteria biomass harvested in exponential growth phase, when half or more of the added dioxane (100 mg/L) was consumed. Total DNA extractions were performed using the UltraClean® Microbial DNA Isolation Kit (MO BIO, Carlsbad, CA, USA) according to the manufacturer’s protocol. The V4 region of the 16S rRNA gene was amplified by PCR using the forward 515F and reverse 806R primers. Sequencing was performed at MR DNA (www.mrdnalab.com, Shallowater, TX, USA) by Illumina MiSeq paired-end sequencing (approximately 2×300 bp as the read length). Sequence data were processed using MR DNA analysis pipeline. Operational taxonomic units (OTUs) were defined by clustering at 3% divergence (97% similarity). Final OTUs were taxonomically classified using BLASTn against the RDPII (http://rdp.cme.msu.edu) and NCBI (www.ncbi.nlm.nih.gov) databases.Previously designed degenerate primers NVC57, NVC58, NVC65 and NVC66 to target conserved regions in the soluble di-iron monooxygenases (SDIMO) alpha subunit gene (groups 3 to 6) were used to examine the presence and diversity of SDIMO genes in these two consortia. A nested PCR strategy was used to increase the PCR product yield. In the first run, the PCR mixture contained 1 µL of NVC65 and NVC58 primer mixture (10 µM), 20 ng of the extracted genomic DNA, 12.5 µL of KAPA HiFi HotStart ReadyMix (2X) (KAPA Biosystems, Wilmington, MA, USA), and nuclease-free water to yield a total volume of 25 µL. PCR was performed in a Bio-Rad Thermal Cycler (Bio-Rad, Hercules, CA, USA) with the following temperature profile: initial denaturation (94°C, 5 min), then 29 amplification cycles (94°C for 30 s, 55°C for 30 s, 72°C for 1 min per kb) and a final extension (72°C for 5 min). The length of the PCR products in the first run was checked by 1% agarose gel and DNA bands of the correct size (1100 bp) were excised and purified. 20 ng of the purified PCR product was used as the DNA template in the second run, with the second set of primers (NVC57 and NVC66). The purified product (420 bp) from the second PCR was sent to MR DNA (www.mrdnalab.com, Shallowater, TX, USA) for Illumina MiSeq paired-end sequencing (approximately 2×300 bp as the read length). Sequence data were processed using MR DNA analysis pipeline. Operational taxonomic units (OTUs) were defined by clustering at 3% divergence (97% similarity). A database including all of the currently reported SDIMO genes on NCBI was created and used to taxonomically classify the final OTUs.

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Two-stage microbial community experimental design.

Publications

Two-stage microbial community experimental design.

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