16S rRNA Amplicon Sequencing of Urban Prokaryotic Communities in the South Bronx River Estuary

ANNOUNCEMENT

With increased urbanization comes the recommendation to monitor disrupted ecological communities and maintain sustainable environments. Much of the biodiversity in New York City, New York, remains to be described, including cryptic prokaryotic communities in the South Bronx River Estuary. Here, 16S rRNA gene amplicons from 33 samples collected at Hunts Point Riverside Park (n_sediment, 9; n_water, 8) and Soundview Park (n_sediment, 8; n_water, 8) are presented. Soundview (40.81N, 73.87W) is among the successful regional oyster restoration sites (1), and Hunts Point (40.82N, 73.88W) is a former illegal dump area being revitalized as a Bronx River Greenway component.

Sampling occurred between August 2015 and September 2016, monthly from May to October during low tide. Soundview samples were collected along the restored oyster reef, an area containing live Crassostrea virginica. Surface water samples were obtained by horizontally submerging a 1-liter autoclaved jar. Core sediment samples weighing approximately 100 g were collected from the surface sediment layer with a polyvinyl chloride pipe and a pallet shovel (2). Samples were placed in a cooler and then a laboratory refrigerator and were processed within 24 h of collection. Water samples were filtered using Whatman cellulose nitrate sterile filters, and a randomly selected 0.25-g soil subsample was used for extraction. DNA was extracted with the PowerWater and PowerSoil kits for water and soil, respectively (Qiagen, USA), and quantified with a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). Samples were stored at −20° C until transfer to MR DNA (Shallowater, TX), where the bacterial V4 hypervariable region segment was amplified with the 16S primers 515F-Y (3) and R806 (4) using the HotStarTaq Plus master mix kit (Qiagen, USA) under standard conditions and controls (5). After agarose gel checks, 3 replicates of the same DNA extract were pooled in equal proportions and purified with calibrated Ampure XP beads (Agencourt Bioscience, USA) (5). A DNA library was created following the Illumina TruSeq protocol, and 16S sequencing was performed using v. 3 chemistry (2 × 250 bp) on the Illumina MiSeq platform.

In total, 4,673,520 raw reads (Hunts Point: n_sediment, 1,130,030, and n_water, 1,200,424; Soundview: n_sediment, 1,363,728, and n_water, 979,338) were sequenced and then processed with QIIME2 using default parameters unless otherwise specified (v. 2019.10) (6). The DADA2 plugin was used to quality filter, denoise, and join paired-end reads (6, 7). Sequences were truncated at 260 bp for quality purposes and trimmed to remove primers. After DADA2 filtering, 2,585,025 (per-sample average, 78,334; standard error, 73,923) or 58% of reads were retained across all samples (Table 1). The QIIME2 naive Bayesian q2 classifier was used for taxonomic classification in comparison to the SILVA 16S rRNA database (8, 9). The amplicon sequence variant (ASV) feature table, taxonomy, and sample metadata were exported to BIOM format for analysis in R (v. 3.6.1) with default parameters (https://www.r-project.org/) using the phyloseq v. 1.29.0 (10), vegan v. 2.5-6 (https://cran.r-project.org/package=vegan), and ggplot2 v. 3.2.1 (11) packages.

After removal of contaminant mitochondria and chloroplast sequences, 508,352 sequences and 16,165 unique ASVs were recovered, and rarefaction analysis showed sufficient sampling depth. The mean diversity indices were higher in sediment (observed ASVs: Hunts Point, 1,283.3, and Soundview, 1,646.5; Shannon diversity: Hunts Point, 6.33, and Soundview, 6.14; Pielou’s evenness: Hunts Point, 0.876, and Soundview, 0.848) than in water samples (observed ASVs: Hunts Point, 612.6, and Soundview, 734.1; Shannon diversity: Hunts Point, 4.69, and Soundview, 5.08; Pielou’s evenness: Hunts Point, 0.732, and Soundview, 0.763). The communities were dominated mostly by the bacterial phyla Proteobacteria, Epsilonbacteraeota, Cyanobacteria, Bacteroidetes, Actinobacteria, and Acidobacteria, but other phyla were present in significant proportions (Fig. 1). These analyses provide a baseline for further characterizing and monitoring the microbial biodiversity in a complex urban estuary.

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FIG 1

Relative abundance bar plot of the top 10 microbial phyla obtained from 16S rRNA sequencing of water (W) and sediment (S) samples at Hunts Point Riverside (HP) Park or Soundview (BRO, SVP) Park. Each color shade represents a different phylum of bacteria or archaea, and sample type is indicated by bars below the graph. Samples collected in 2016 have “16” included in the name, while all other samples were collected in 2015. All organisms belong to the domain Bacteria, except for the Nanoarchaeota, which is classified in Archaea.