Effect of PEX PCR Stage “A” annealing temperature on observed microbial community structure and primer utilization patterns.

<p>Non-metric multidimensional scaling (NMDS) plot of fecal microbiome, performed at the taxonomic level of family and based on Bray-Curtis similarity (2D stress = 0.05). Samples were rarefied to 1,250 sequences per sample and no transformation was applied. The analysis is based on a single genomic DNA sample, with PEX PCR stage “A” annealing performed at 30°C (down-facing triangles), 35°C (open circles), 40°C (diamonds), 45°C (up-facing triangles), 50°C (closed circles) and 55°C (squares). Symbols are color-coded by the diversity (Shannon Index) of reverse primers (<i>i</i>.<i>e</i>. 806R) utilized in annealing and elongation during stage “A” of PEX PCR. Maximum possible Shannon index for 18 primers in the primer pool is 2.89. Vectors indicate taxa with Pearson correlation of >0.8 with MDS1 and MDS2 axes.</p

Temperature gradient analysis of the PEX PCR and TAS methods using mock community DNA.

<p>The relative abundance of reads mapping to the each of the four target templates (Mock A, Mock B, Mock C and Mock D) is shown for each temperature. The error bars represent standard deviation associated with two to four replicates per sample. <b>(A)</b> Results from PEX PCR and <b>(B)</b> Results from TAS PCR.</p

Schematic of Targeted amplicon sequencing (TAS) and Polymerase Exonuclease (PEX) PCR methods.

<p>Schematic of Targeted amplicon sequencing (TAS) and Polymerase Exonuclease (PEX) PCR methods.</p

Effect of PEX PCR and exonuclease treatment on observed microbial community structure and primer utilization patterns.

<p>Non-metric multidimensional scaling (NMDS) plot of lake sediment microbiome, performed at the taxonomic level of family and based on Bray-Curtis similarity (2D stress = 0.02). Samples were rarefied to 35,500 sequences per sample and no transformation was applied. All reactions were performed with an annealing temperature of 45°C, using PEX PCR with exonuclease (squares), PEX PCR without exonuclease (circles), and TAS PCR (triangles). Symbols are color-coded by the diversity (Shannon Index) of reverse primers (<i>i</i>.<i>e</i>. 806R) detected in the sequences. Maximum possible Shannon index for 18 primers in the primer pool is 2.89. Small, but significant differences in the observed family-level Shannon index (F-SI) were observed between PEX PCRs (with and without exonuclease treatment) and TAS PCRs using a two-tailed ttest (p<0.05).</p

Types and abundance of DNA fragments found in PCR.

<p><b>(A)</b> Template DNA fragments (containing strands “A” and “B”) are added to PCR reactions and are conserved throughout the reaction. Fragments “A” and “B” serve as templates for copying in each cycle, with hybrid molecules “C” and “D” produced in a linear fashion each cycle. In cycles two and above, “C” and “D” are copied, creating hybrid molecules “E” and “F” in a linear fashion. In cycles three and above, the “E” and “F” fragments generated in prior cycles are copied into inverse complement fragments “F” and “E”, respectively, in an exponential fashion. Red boxes indicate ‘natural’ primer annealing to genomic DNA template or copy of gDNA template. Green boxes indicate ‘artificial’ primer annealing to primer sites that are copies of oligonucleotide primers added to the PCR mixture, and incorporated during previous cycles. <b>(B)</b> The relative abundance of “E” and “F” fragments generated by ‘natural’ template-primer interactions (“C”,”D” → “E”,”F”; shown as solid squares) and by artificial template-primer interactions (“E”,”F” → “F”,”E”; shown as open circles) varies by cycle. At the end of cycle two, all “E” and “F” fragments have been generated only by ‘natural’ template-primer interactions.</p

Relative abundance of mock DNA templates observed in sequencing of TAS and PEX PCR method reactions.

<p>The error bars represent standard deviation associated with two replicates per sample.</p

Beta-diversity of bacterial communities present in leaves of <i>S. purpurea</i> from NY and FL sampling locations using Bray-Curtis dissimilarity.

<p>MDS plots comparing bacterial community structure within sampling sites and among leaves were generated based on organismal classification based on colony morphology of culturable bacteria (A) and 16S rRNA gene sequences, identified at the genus-level using the RDP classifier (B). Data were based on square-root transformed Bray-Curtis similarity. Each symbol represents the bacterial community in one pitcher plant leaf (FL = Florida; NY = New York).</p

The Bacterial Composition within the Sarracenia purpurea Model System: Local Scale Differences and the Relationship with the Other Members of the Food Web

The leaves of the carnivorous pitcher plant, Sarracenia purpurea, contain a microscopic aquatic food web that is considered a model system in ecological research. The species identity of the intermediate and top trophic level of this food web, as well the detritivore midge, are highly similar across the native geographic range of S. purpurea and, in some cases, appear to have co-evolved with the plant. However, until recently, the identity, geographic variation, and diversity of the bacteria in the bottom trophic level of this food web have remained largely unknown. This study investigated bacterial community composition inside the leaves of S. purpurea to address: 1) variation in bacterial communities at the beginning of succession at the local scale in different areas of the plant’s native geographic range (southern and mid-regional sites) and 2) the impacts of bacterial consumers and other members of the aquatic food web (i.e., insects) on bacterial community structure. Communities from six leaves (one leaf per plant) from New York and Florida study sites were analyzed using 16S ribosomal RNA gene cloning. Each pitcher within each site had a distinct community; however, there was more overlap in bacterial composition within each site than when communities were compared across sites. In contrast, the identity of protozoans and metazoans in this community were similar in species identity both within a site and between the two sites, but abundances differed. Our results indicate that, at least during the beginning of succession, there is no strong selection for bacterial taxa and that there is no core group of bacteria required by the plant to start the decomposition of trapped insects. Co-evolution between the plant and bacteria appears to not have occurred as it has for other members of this community

Community composition of Florida phytotelmata.

1<p>Other invertebrates observed within leaf water.</p>2<p>Density and Richness determined in a 0.1 ml aliquot of leaf water.</p>3<p>Density determined in a 0.1 ml aliquot of leaf water after a 10⁻⁴ dilution.</p><p>Community data (with the exception of bacterial sequences) collected in all 15 pitchers sampled in the Florida bog. The first six samples were the samples also used for the RDP classification analysis.</p

Beta-diversity of bacterial communities present in leaves of <i>S. purpurea</i> from NY and FL sampling locations using the Unifrac metric.

<p>Bacterial community structure was assessed by sequencing 16S rRNA genes as described in the text. A phylogenetic tree was generated by inserting partial gene sequences recovered in this study into a tree based on near-full length sequences, implemented within the software package ARB. The phylogenetic tree was analyzed using the software package Unifrac, and a pair-wise distance matrix was generated for comparison of the bacterial community in each leaf. This matrix was used to generate a MDS plot, demonstrating distinct bacterial communities in FL and NY leaves.</p