Computational protocol: Bromeliad Catchments as Habitats for Methanogenesis in Tropical Rainforest Canopies

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Protocol publication

[…] Freshly collected bromeliad tank water (0.5 ml, including debris) was spun at 15,000 × g for 10 min and the resulting pellet was either extracted for total nucleic acids or preserved in 0.5–1.0 ml RNAlater (Ambion, Inc.). DNA was extracted using the Power Soil DNA extraction kit (MoBio Laboratories, Inc., Carlsbad, CA, USA), modified by two initial 5–10 min incubations at 65°C, one in the presence of solution S1, followed by 5–10 min vortexing. The remainder of the extraction procedure was carried out according to the manufacturer’s instructions, with the exception of a 4°C incubation in IRS solution (5 min) between solutions S2 and S3 to increase DNA yield and inhibitor removal. SSU (16S) rRNA was amplified by polymerase chain reaction (PCR) from extracted DNA, using the archaea-specific primer pair 1F/1100R (1F, 5′-TCYGKTTGATCCYGSCRGAG-3′; 1100R, 5′-TGGGTCTCGCTCGTTG-3′; Hales et al., ), the methanogen-specific primer pair 355F/1068R (355F, 5′-CAGGCGCGAAAACTTTAC-3′; 1068R, 5′-ATGCTTCACAGTACGAAC-3′; Banning et al., ), or a combination of 355F/1100R (Table ). Thermal cycling conditions were as described, with an annealing temperature of 52°C, for 25 cycles (Hales et al., ; Banning et al., ). No difference in relative abundance and types of methanogens was detected between those recovered using the two main primer pairs (see Wg104; Table ).Presence/absence of methanogens was judged by electrophoretic separation and visualization of PCR products produced by the 355F/1068R primer set. For seven bromeliads, one artificial catchment, and one soil sample, PCR-amplified bacterial 16S rRNA genes were used for clone library construction. PCR products were pooled in triplicate prior to ligation. Transformants (30–48 clones analyzed for each bromeliad library; Table ) were screened directly for the presence of inserts using M13F/R vector primers (8 min initial denaturation). M13 amplicons were cleaned prior to sequencing with MultiScreen HTS plates (Millipore Corporation, Bedford, MA, USA). Sequencing reactions were performed using the Genome Lab DTCS Quick Start Kit (Beckman Coulter, Fullerton, CA, USA), precipitated according to the manufacturer’s instructions, and run on a CEQ 8800 Genetic Analysis System (Beckman Coulter, Fullerton, CA, USA). Representative ribotypes, based on 97% sequence similarity, were selected for near full-length sequencing (Figure ).Sequences were assembled and edited using Sequencher v4.10.1 (Gene Codes Corporation). Initial sequence homology searches were performed using BLASTn (NCBI) and the Ribosomal Database Project classifier. Our 16S rRNA sequences along with additional sequences obtained from GenBank were compiled in ARB, after initial alignment using the SILVA Aligner function, with subsequent manual refinements (Ludwig et al., ; Preusse et al., ). For near full-length representatives and closest relatives, neighbor-joining (NJ) analysis was conducted with Felsenstein distance correction. In some cases, partial sequences recovered in our study were added to the NJ tree in ARB via parsimony insertion within a tree of longer sequences. NJ analysis was performed with 2000 bootstrap replicates to assign confidence levels to nodes, shown in Figure , if >70% confidence (PAUP*4.0b10; Swofford, ). Sequences obtained in this study have been deposited in the GenBank database under accession numbers JN810747–JN810784 (archaeal 16S rRNA ribotypes) and JN810785–JN810789 (mcrA genes). […]

Pipeline specifications

Software tools Sequencher, BLASTN
Application Phylogenetics
Chemicals Carbon, Methane