Miseq pe300 sequencer

The PCR products were isolated using a 2% agarose gel, purified and quantified. A PE 2^*300 library was constructed using the purified amplification fragments according to the standard procedure of the TruSeqTM DNA Sample Prep Kit (Illumina, San Diego, USA). Finally, the DNAs were sequenced using the Miseq PE300 sequencer (Illumina, San Diego, USA).

fRuSau02 DNA was isolated from crude phage lysate with Invisorb Spin Virus DNA Mini Kit (Stratec Biomedical, Birkenfeld, Germany). Sequencing was performed at the Institute for Molecular Medicine Finland (FIMM) Technology Centre Sequencing Unit [24 ]. For next-generation sequencing, the DNA library was constructed with Nextera sample prep kit (Illumina, San Diego, CA, USA). Paired-end sequencing was done using Illumina MiSeq PE300 sequencer (Illumina, San Diego, CA, USA) with the read length of 300 nucleotides. TheA5 (Andrew And Aaron’s Awesome Assembly)-miseq integrated pipeline for de novo assembly of microbial genomes was used to obtain the genome sequence [25 (link)]. fRuSau02 sequence was submitted to GenBank with accession number MF398190.

DNA from 62 stool samples was sequenced and primers (pre-primer sequence 5, -ACTCCTACGGGAGGCAGCA-3, post-primer sequence 5, -GGACTACHVGGGTWTCTAAT-3) were used to amplify the V3 and V4 regions of the 16S rRNA gene. After sequencing library preparation, the MiSeq Reagent Kit V3 (Illumina, USA) was used to perform paired-end sequencing on a MiSeqPE300 sequencer (Illumina, USA). Raw sequences were quality-controlled, denoised, spliced, and dechimerized using QIIME2 to create a signature sequence (ASV) abundance table. Species annotation was performed on each signature sequence, based on the Greengenes database using QIIME2 software with default parameters.

Using the Fast DNA SPIN Extraction Kit (MP Biomedicals; Santa Ana, CA, USA), genomic DNA was extracted from 0.5 g of soil following the manufacturer’s guidelines. DNA quantity and quality were first detected by agarose gel electrophoresis (1%) and then detected by using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific; Waltham, MA, USA). The fungal ITS1 region was targeted using the ITS1F (5′-CTT GGT CAT TTA GAG GAA GTA A-3′) and ITS2 (5′-GCT GCG TTC TTC ATC GAT GC-3′) primer set [29 ] (White et al., 1990). A 7 bp sample-specific DNA barcode was incorporated into the primers for each unique sample to allow for multiplex sequencing. All PCR procedures were conducted in two steps. First, PCR was conducted in triplicate (i.e., three independent 25 µL reactions per DNA sample). Each reaction contained 12.5 µL of 5x PCR mixed reaction buffer, 1 µL (10 µM) of forward and reverse primers, 2 µL of DNA template, and 9.5 µL of ddH2O. The PCR procedure consisted of a 10 min denaturation step at 96 °C followed by 32 cycles at 95 °C for 50 s, 55 °C for 60 s, and 72 °C for 60 s, and finally 72 °C for 10 min. The PCR products were purified using the TAKARA DNA Gel Extraction Kit (TAKARA Biosciences). Afterward, the PCR products were pooled at equal proportions and sequenced conducted by Illumina MiSeq PE300 sequencer.

The colonic bacterial DNA was isolated with the E.Z.N.A.^® soil kit (Omega Bio-tek, USA) according to the instructions. DNA concentration and purity were investigated by NanoDrop2000. The V3-V4 regions were amplified using specific primers, 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). PCR products were purified with AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, USA). Sequencing of the TruSeq DNA libraries was done on a Miseq PE300 sequencer (Illumina, USA) following the manufacturer’s recommendations. Trimmomatic software was used for quality control and FLASH (Fast Length Adjustment of SHort reads) software was used for splicing. The taxonomic unit was screened for further annotation (Korpela et al., 2016 (link)).

Approximately 0.5 g of soil was weighed before adding exogenous Hg and after aging. Thereafter, DNA was extracted using a Fast DNA SPIN Kit according to the manufacturer’s instructions. The extracted soil DNA was subsequently amplified by PCR, using universal primers with different TAG tags. The cut gel was purified using a NanoDrop^® (Thermo Fisher Scientific, Waltham, MA, USA). The DNA concentration was determined using an ND-2000 UV-Vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and the purified DNA was sequenced using a MiSeq PE 300 sequencer (Illumina Inc., San Diego, CA, USA) from Shanghai Meiji Biomedical Technology Co., Ltd. (Shanghai, China) After sequencing, low-quality sequences were removed, effective sequences were distinguished, and the sequence direction was adjusted according to the barcode and primer sequences.