title: A Microbial Signature Approach to Identify Fecal Pollution in the Waters Off an Urbanized Coast of Lake Michigan
design: Water quality is impacted significantly by urbanization. The delivery of increased nutrient loads to waterways is a primary characteristic of this land use change. Despite the recognized effects of nutrient loading on aquatic systems, the influence of urbanization on the bacterial community composition of these systems is not understood. We used massively-parallel sequencing of bacterial 16S rRNA genes to examine the bacterial assemblages in transect samples spanning the heavily urban impacted rivers and harbor of Milwaukee, WI USA to the relatively un-impacted waters of Lake Michigan. From these data, we examined the source, the conditions promoting enhanced delivery, and the persistence of fecal bacteria and urban-infrastructure bacteria in the waters surrounding the city of Milwaukee. We also examined whether the conditions in urban waterways impact the composition of common freshwater bacterial lineages and assemblage characteristics of the microbial communities present.    We constructed amplicon libraries for the V6 through V4 hypervariable domains (amplification in reverse DNA strand orientation). To set up the amplification procedure, amplification fusion primers contained either the A or B 454 Titanium amplicon adapter for GS-FLX sequencing (Roche Diagnostics; Indianapolis, IN, USA) followed by a 5-nt multiplex identifier (MID) and ending with a 16S-specific sequence. The 16S sequences used were 518 F, 5′CCAGCAGCYGCGGTAAN and 1064R, 5′CGACRRCCATGCANCACCT. The PCR mixture contained 1X Platinum HiFi Taq polymerase buffer, 1.6 units Platinum HiFi polymerase (Life Technologies, Carlsbad, CA, USA), 3.7 mM MgSO4, 200 μM dNTPs (PurePeak polymerization mix, ThermoFisher, E. Providence, RI, USA), and 50 nM combined primers. Five to 25 ng of sample DNAwas added to the PCR master mix to a final volume of 100 μl, and this was divided into three replicate 33-μl reactions. We included a no-template negative control for each MID. PCR conditions included an initial denaturation at 94 °C for 3 min; 30 cycles of 94 °C for 30 s, 60 °C for 45 s, and 72 °C for 1 min; and a final extension at 72 °C for 2 min using an Applied Biosystems 2720 or 9700 cycler (Life Technologies). We cleaned the reaction and removed products under 300 bp using Ampure beads at 0.75°— volume (Beckman Coulter, Brea, CA, USA). The final products were resuspended in 100 μl of 10 mM Tris-EDTA. The emulsion PCR, enrichment, and sequencing were done according to current Roche Titanium amplicon sequencing protocols (Lib-A emPCR reagents, XLR sequencing reagents, two region PicoTitre plate). Image processing and signal calling were done using the Roche amplicon-processing pipeline (version 2.53) with recursive phase correction algorithm to maximize the number of long reads. Following sequencing, we quality-filtered the V6V4 reads by removing reads without exact matches to the 1064R sequence and the MID, reads containing ambiguous bases, and reads that lacked the conserved sequence 5′- TGGGCGTAAAG-3′ (position 565 F in E. coli) allowing two mismatches, or that had a quality score <30. Sequence reads were trimmed at the same evolutionarily homologous position proximal to the 565 F conserved sequence. UChime using both a de novo and reference database (ChimerSlayer GOLD) eliminated chimeras. The Global Alignment for Sequence Taxonomy (GAST) program assigned taxonomy.