Phix control dna

Of the 85 tap water samples collected, 67 yielded sufficient DNA for high-throughput sequence analysis. Bacterial profiles were determined by amplification of 16S rRNA genes and sequence analysis following previously described methods [28 (link),29 (link)]. Amplicons were generated using primers that target the V1-V2 region (27F/338R). PCR products were normalized using agarose gel densitometry and pooled. The pooled amplicons were lyophilized, purified (Montage), concentrated using a DNA Clean and Concentrator kit (Zymo Research, USA) and adjusted to 4 nM. The pooled DNA was denatured, then diluted to 15 pM and spiked with 25% of the Illumina Phi X control DNA prior to loading the sequencer. Illumina paired-end sequencing was performed at the University of Colorado at Denver on the MiSeq platform with version v2.3.0.8 of the MiSeq Control Software and version v2.3.32 of MiSeq Reporter, using a 600-cycle v3 (2x300) reagent kit.

16S rDNA libraries were quantified with KAPA DNA Library Quantification Kit (KAPA Biosystems, Wilmington, MA, USA). Reactions prepared according to the manufacturer’s protocol were performed on LightCycler 480 System (Roche Applied Science, Penzberg, Upper Bavaria, Germany). The libraries were mixed in equimolar ratio and diluted to obtain one 6 pM sequencing library with 10% PhiX Control DNA (Illumina, San Diego, CA, USA). Sequencing was performed on Illumina MiSeq using MiSeq Reagent Kit v3 (600 cycles) (Illumina, San Diego, CA, USA). HPLC-purified custom sequencing primers were mixed with Illumina ones. Demultiplexing of indexed reads was made according to Illumina standard protocol.

Bacterial profiles were determined by broad-range amplification and sequence analysis of 16S rRNA genes following our previously described methods (Hara et al., 2012 (link); Markle et al., 2013 (link)). Amplicons were generated using primers that target approximately 300 base pairs of the V1/V2 variable region of the 16S rRNA gene. Each DNA was amplified in triplicate along with a barcode specific negative PCR control. PCR reactions contained 1X HotMaster Mix (5Prime), 150 nM each PCR primer and template in a reaction volume of 25 µl. Cycling conditions were 94 °C denaturation for 120 s followed by 30 cycles of 95 °C 20 s, 52 °C 20 s and 65 °C 60 s. Amplification was confirmed using agarose gel electrophoresis. None of the negative PCR controls showed evidence of amplification. PCR products were normalized based on agarose gel densitometry, pooled, lyophilized, purified and concentrated using a DNA Clean and Concentrator Kit (Zymo, Irvine, CA, USA). Pooled amplicons were quantified using Qubit Fluorometer 2.0 (Invitrogen, Carlsbad, CA, USA). The pool was diluted to 4 nM and denatured with 0.2 N NaOH at room temperature. The denatured DNA was diluted to 15 pM and spiked with 10% of the Illumina PhiX control DNA prior to loading the sequencer. Illumina paired-end sequencing was performed on the MiSeq using a 500 cycle version 2 reagent kit.

PCR was performed using universal primers flanking the V4 region of the bacterial 16S rRNA gene [13 (link)]. A total of 25 ng DNA, 0.4 μM each primer, 12.5 μl 2X KAPA HiFi HotStart ReadyMix (Kapa Biosystems, Wilmington, US), and water to 25 μl were used for one reaction per sample. Cycling conditions began with an initial denaturation of 95°C for 3 minutes followed by 25 cycles of 95 °C for 30 seconds, 55 °C for 30 seconds, and 72 °C for 30 seconds, with a final extension at 72 °C for 5 minutes. PCR products were purified by gel extraction from a 1.0% low-melt agarose gel using the ZR-96 Zymoclean Gel DNA Recovery Kit (Zymo Research Corp, Irvine, US). Samples were quantified using a Qubit Fluorometer 2.0 (Invitrogen, Waltham, US) and pooled at equimolar concentrations. The pool plus 5% PhiX control DNA (Illumina, Inc, San Diego, US) was sequenced with the MiSeq 2x250 v2 reagent kit (Illumina, Inc, San Diego, US) using custom sequencing primers [13 (link)].