Ampure xp

For the annealing blocker experiments, samples were normalized by quantifying the indexing PCR reactions using a Quant-iT PicoGreen dsDNA Assay Kit (ThermoFisher Scientific), normalizing samples for each amplicon (V4 and V9) to an equal concentration, pooling 3 µl of each normalized sample, purifying and concentrating with a 1x AmPureXP (Beckman Coulter) clean up, and eluting in 25 µl of Qiagen buffer EB. For the PNA blocker experiments, indexing PCR reactions were purified and normalized using a SequalPrep Normalization Plate Kit (ThermoFisher Scientific). 10 µl of each sample was pooled (V4 and V9 were pooled separately, due to the different sizes of these amplicons) and the pools were purified and concentrated with a 1x AmPureXP (Beckman Coulter) clean up, followed by elution in 25 µl of Qiagen buffer EB.
The concentrations of the amplicon pools were determined using a Quant-iT PicoGreen dsDNA Assay Kit (ThermoFisher Scientific) and amplicon sizes were verified on an Agilent Bioanalyzer High Sensitivity Chip. The V4 and V9 amplicon pools were independently diluted down to a 2 nM concentration in Qiagen EB buffer, and mixed at a 1:1 ratio.

A total of 250 ng of total RNA were taken for library preparation using Illumina TruSeq Stranded Total RNA Library Prep Gold (Illumina, Cat. No. 20020598) as per manufacturer’s reference guide (1000000040499 v00). The ribosomal RNA (both cytoplasmic and mitochondrial) were first removed using biotinylated target specific oligos followed by fragmentation. First strand cDNA synthesis was carried out from the cleaved RNA fragments using reverse transcriptase and random primers, followed by second strand synthesis using DNA polymerase 1 and RNase H. This double stranded cDNA has been purified using AMPure XP (Beckman Coulter, A63881) followed by adenylation of 3ʹ blunt end of the double stranded cDNA. Each library was tagged with unique indexes which were then enriched by PCR amplification. Subsequently, the final library was purified using AMPure XP (Beckman Coulter, A63881) and quality of libraries checked using Agilent 2100 Bioanalyzer. Final loading concentration of 650 pM was used for sequencing, performed on the NextSeq 2000, with paired end 2 × 151 read length.

DNA was first amplified with AOA and AOB amoA primers (Table S1 in File S1, [50] (link), [51] (link)) under conditions presented in Table S7 and S8 in File S1. The PCR products were diluted 1∶10 and used as template for a second PCR using the barcoded primers (Table S5, Table S6 and Table S9 in File S1). Twelve bar-coded primers were generated, six for archaeal amoA and six for bacterial amoA, allowing sequencing of the amoA genes of six independent samples in 1/16 of a 454 sequencing run. The two-step PCR prevents amplification biases and increases reproducibility [52] (link). Per DNA isolation, three PCR reactions were conducted and at the end all PCR products (triplicate PCR runs × triplicate DNA isolations) per sample were mixed and used as one sample for pyrosequencing. The samples were purified with AMPureXP (Beckman-Coulter, Inc., Indianapolis, IN, USA). The samples were quantified with PicoGreen assay, diluted, pooled, and purified again with AMPureXP (Beckman-Coulter, Inc., Indianapolis, IN, USA). The concentration in the pooled samples was determined using KAPA qPCR (KAPA Biosystems, Woburn MA, USA). For the first library 0.5 copies per bead and for the second library 2 copies per bead were sequenced on the Roche GS FLX system at the Plant-Microbe Genomics Facility at The Ohio State University (Columbus, Ohio, USA).

We prepared amplicon libraries, targeting the ITS1 region, using ITS1F and ITS2 primers (35 (link), 36 (link)). Amplicon were generated by PCR using a 96-well thermal cycler in the following conditions: 95°C for 3 min, 25 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 30 s, and 72°C for 5 min, and cooling at 4°C. Amplicons were purified with AMPure XP (Beckman Coulter, USA) as described in the 16S Metagenomic Sequencing Library Preparation guide (37 ). Adapter were attached using Nextera XT Index Kit (Illumina, France) and the index PCRs were performed in the following conditions: 95°C for 3 min, eight cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 30 s, and 72°C for 5 min, and cooling at 4°C. Barcoded PCR products were purified with AMPure XP (Beckman Coulter, USA) and verified and quantified on a Bioanalyzer DNA 1000 chip (Agilent, USA). Samples were normalized at 4 nM and pooled into a library, using 5 μL of each diluted sample. A PhiX sequencing control was prepared following the manufacturer’s instructions. The libraries were sequenced in 300-bp paired-end using the MiSeq reagent kit V3 on Illumina MiSeq platform (Illumina, Evry, France).

17.5 μL of 2X Q5 polymerase (NEB) was added to 10μL of purified DNA with 2.5 μL of i7 Nextera index primer, 2.5 μL L of i5 Nextera index primer, 0.5 μL of ILMN1 primer (50 μM), 0.5 μL of ILMN2 primer (50 μM), 1 μL 5-methyl-dCTP (10 μM) and 0.5 μL H
₂O. After an initial 72°C for 3 minutes and 98°C for 30 s, the library was amplified for 12 cycles of 98°C for 10 s, 63°C for 30 s, 72°C for 1 minute and a 10°C hold. Use of methylated nucleotides for PCR decontamination is described previously
^{26 (link),
27 (link)}. PCR samples were purified by mixing 52.5 μL of Agencourt Ampure XP into the PCR reaction. The samples were placed on a magnet for 15 minutes until the beads cleared and the supernatant could be removed. Beads were washed twice with 200 μL of 70% EtOH. Beads were left for 10 minute to air dry and then eluted in 25 μL of 10 mM Tris-HCl. 5 μL of each PCR product was pooled and quantified with a Qubit (Thermo) for proper dilution onto MiSeq version 2 chemistry according to the manufacturers’ instructions. 2×150 bp reads were selected to obtain maximal ITS2 sequence information.

After HCMV-BAC preparation, amplicons were purified using magnetic beads (Agencourt AMPure XP) and fragmented using the Ion Xpress Plus DNA Fragment Library Preparation kit (Life Technologies). Barcodes adapters were ligated to fragment ends and 250 bp fragments were collected. The library was PCR amplified, then sequenced on the Ion Proton with the Ion Sequencing kit (Life Technologies). Bases callings were performed with Torrent Suite Software version 5.0.2. Mutations were obtained using Torrent Variant Caller using Somatic variant frequency and AD169_ATCC as reference. Mutations were then filtered against reference (Wild-type HCMV-BAC) using vcftools version 0.1.13.