Mobio powersoil dna isolation kit

At the end of the incubation experiment, soil samples for molecular analyses were immediately stored at −80°C before the aforementioned analysis. DNA was extracted from 0.3 g of frozen soils using a Mo Bio PowerSoil DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, CA, United States) according to the manufacturer’s protocol. The concentration and purity of the extracted DNA were determined with a spectrophotometer at 260 nm (NanoDrop Technologies, United States). All extracted soil DNA samples were stored at −80°C before analysis. Quantitative polymerase chain reaction (qPCR) was used to estimate the abundance of nitrification and denitrification functional genes (amoA, nirK, nirS, and nosZ) using a real-time PCR detection system. The PCR primers used in this study are listed in Supplementary Table 1. The 10-fold serially diluted plasmids carrying each target gene were subjected to real-time PCR assays in triplicate to generate a standard curve. The qPCR efficiencies for AOA-amoA, AOB-amoA, nirK, nirS, and nosZ were 0.85, 0.94, 0.96, 0.91, and 0.87, respectively. The corresponding determination coefficients of the standard curve for AOA-amoA, AOB-amoA, nirK, nirS, and nosZ were 0.998, 0.993, 0.990, 0.999, and 0.996, respectively.

Eighty milliliters of the water enriched with microorganisms used for the biodegradation tests was filtered (0.2μm porosity, cellulose nitrate filters, Sartorius Stedim). The filters were then ground in liquid nitrogen to a fine powder, and DNA was extracted using the Mobio-Power Soil DNA isolation kit (Mobio Laboratories, Carlsbad, CA) in line with the supplier’s recommendations. The genomic DNA was sent to a commercial company (MR DNA, Shallowater, Texas, United States). Following the amplification of the V4 hypervariable region of the 16S rRNA gene (PCR primers 515/806), the amplicons were sequenced via MiSeq 2 × 300bp sequencing (Illumina, California, United States) in line with the supplier’s recommendations. The sequences were converted and demultiplexed with QIIME1. The sequences were analyzed using the software package DADA2 (Callahan et al., 2016 (link)) in the QIIME2 pipeline. With this package, the sequences were treated by quality filtering, merging, dereplicating, and removing chimeras to determine amplicon sequence variants (ASVs). Taxonomy was assigned using VSearch (Rognes et al., 2016 (link)) against the SILVA SSU 138 NR database (Quast et al., 2013 (link)) without uncultured/environmental sequences. The raw data were deposited in the NCBI SRA under bioproject ID PRJNA739070.

The column sediments were divided into three layers, the surficial layer N1 (sediment depth 1–5 cm), the mid-layer N2 (5–10 cm), and the deep layer N3 (10–15 cm). According to the standard manufacturer protocol, about 10 g of sediment sample were used to extract DNA for each sediment layer, using the MoBio PowerSoil DNA Isolation kit (MO BIO Laboratories, Carlsbad, CA, USA). Three DNA extractions were performed on every sediment sample and were then mixed for Illumina library construction, which minimizes the variation between different extractions. Two Illumina libraries were built for the extraction mixture of each sediment layer, and two separate runs sequenced each library. The first three Illumina libraries of the three samples were sequenced in one Hiseq run and the second sequence for another run to overcome the variation between different sequencing runs. Notably, PCR amplification was limited to 12 cycles for each Illumina library. The quality and quantity of genomic DNA were examined by an Agilent Bioanalyzer 2100 (Agilent Technologies, Redwood City, CA, USA). Metagenomic shotgun sequencing was performed on the Hiseq Xten instruments (Illumina, San Diego, CA, USA), with 2 × 150 bp paired-end reads.

Total soil DNA was isolated from 0.5 g of initial material using the MoBio PowerSoil DNA isolation kit (MoBio Laboratories, Carlsbad, CA, USA). The manufacturer’s protocol was slightly modified by the addition of glass beads (diameter 0.1 mm; 0.25 g) to the soil slurries followed by three cycles of bead beating (mini-bead beater, BioSpec Products, Bartlesville, OK, USA) for 60 s. Obtained DNA samples were quantified using the Quant-iT PicoGreen dsDNA assay kit (Invitrogen, Carlsbad, CA, USA) on a TECAN infinite M200 Pro (Maennedorf, Switzerland) plate reader reading at 485 nm excitation and 530 nm emission. All samples were standardized at equal concentrations for further analysis.

DNA was extracted from 0.3 g of soil using the MoBio PowerSoil™ DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, United States) following the instructions of the manufacturer. The quantity and quality of the extracted DNA were determined using a DeNovix DS-11 spectrophotometer (DeNovix, Wilmington, DE, United States).
The primers of 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGG TWTCTAAT-3′) were used to amplify the V3–V4 region of bacterial 16S rRNA gene (Zhang et al., 2015 ), and the fungal internal transcribed spacers (ITSs) were amplified using the eukaryote-specific primers ITS5-1737F (5′-GGAAGTAAAAGTCGTAACAAGG-3′) and ITS2-2043R (5′-GCTGCGTTCTTCATCGATGC-3′) (White et al., 1990 (link)). PCR reactions were carried out with 15 μl of Phusion^® High-Fidelity PCR Master Mix (New England Biolabs, Ipswich, United Kingdom), 0.2 μM forward primer, 0.2 μM reverse primer, and about 10 ng of template DNA. The PCR products were purified with Qiagen Gel Extraction Kit (Qiagen, Hilden, Germany). The high-throughput sequencing was performed on an Illumina NovaSeq platform provided by Novogene Bioinformatics Technology Co., Ltd., Beijing, China.

Bacterial genomic DNA was extracted from buccal and stool specimens using the MO BIO PowerSoil DNA Isolation Kit (MO BIO Laboratories). The 16S rRNA V4 region was PCR amplified and sequenced on the Illumina MiSeq platform using a 2 × 250-bp paired-end protocol adapted from the Human Microbiome Project (HMP) methods [16 (link), 27 (link)]. All samples from the same patient and site were processed and sequenced together to minimize batching issues. Amplification primers contained adapters for MiSeq sequencing and single-index barcodes resulting in PCR products that were pooled and sequenced directly. Read pairs were de-multiplexed based on barcodes and merged using USEARCH v7.0.100. 16S rRNA gene sequences were allocated to specific operational taxonomic units using a UPARSE pipeline and aligned to the V4 region within the SILVA SSURef_NR99_119 database [28 (link)]. Analysis of microbiome communities was performed in R (R Core Team 2015, version 3.2.2, http://www.R-project.org), using phyloseq [29 ] to calculate α- and β-diversity metrics. The Shannon Diversity Index (SDI) was used for α-diversity calculations, and weighted and unweighted UniFrac for β-diversity distances [30 (link)]. The 16S V3–V4 region HMP sequencing reads were obtained from http://hmpdacc.org/HMQCP, trimmed to match the region amplified by this study, and processed identically to AML patient samples.