Mixed Liquor of a SBR treating digested swine waste by nitration/anammox
The V4 region of the 16S rRNA gene was amplified using PCR primers 515F/806R with barcode on the forward primer. The PCR reaction used HotStarTaq Plus MasterMix Kit (Qiagen, Valencia, CA, USA) under the following conditions: 94°C for 3 minutes, followed by 28 cycles of 94°C for 30 seconds, 53°C for 40 seconds and 72°C for 1 minute, with a final elongation step at 72°C for 5 minutes. PCR products were confirmed on a 2% agarose gel. Multiple samples were pooled in equal proportions based on molecular weight and DNA concentration. Pooled samples were subsequently purified using calibrated Ampure XP beads. The pooled and purified PCR product was used to prepare a DNA library by following the Illumina TruSeq DNA library preparation protocol. Amplification of sample DNA and sequencing were performed at MR DNA (www.mrdnalab.com, Shallowater, TX, USA) on an Illumina MiSeq following the manufacturer?s guidelines. Forward and reverse reads were joined, exact barcode matches culled from the full dataset and oriented to the 5`?3` direction at MR DNA labs. Libraries were further processed with mothur v.1.35.1 (Kozich et al 2013) and analyses followed the ?Schloss MiSeq Standard Operating Procedure (SOP)? (http://www.mothur.org/wiki/MiSeq_SOP). Data processing and clean-up included demultiplexing the run, removing barcodes and primers, discarding short (less than 227 bp) and long (greater than 275 bp) reads, and discarding reads that contained ambiguous base calls or long (greater than 8) homopolymer runs. Only reads with an average quality score >25 were retained. Sequences were checked for chimeras using the UCHIME implementation in mothur and chimeric sequences removed from analysis. The Greengenes database (DeSantis et al 2006) was used for classification of representative sequences and Archaea were retained in the final dataset. Sequences were classified into operational taxonomic units (OTUs) based on 97% homology. OTUs that comprised a single read were removed from downstream analysis.
SUB1156877
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