Miseq high throughput sequencing

The community compositions and metabolic activities of microbiota in different sections of the purification system were investigated using Illumina Miseq high-throughput sequencing and Biolog microplate techniques. Microbial samples were collected from culture pond water, biofilms on the artificial substrates, ASFP outfall water, biofilms on the I. aquatica rhizosphere in the AFBFP, AFBFP outfall water, biofilms on the brushes, BrFP, outfall water, and water from the storage pond on August 6, 2014. Each biofilm sample was collected from three subsites on the filter and mixed. Approximately 50 g (wet weight) of the biofilms from each site including the artificial substrate, the I. aquatica rhizosphere, and the brush was cut into fragments and placed in 500-mL sterile plastic sampling bottles. Three replicate 1-L samples were collected from each location, totaling 24 samples altogether. These were stored at 4°C and transported to the laboratory for Biolog plate analysis and DNA extraction.

The bacterial communities in the fecal samples were investigated by Illumina MiSeq high-throughput sequencing.
The V3 and V4 regions of the 16S rDNA gene were selected for PCR. The primers were barcoded-338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′; H, W, and V were degenerate bases; H represented A, T or C; V represented G, A or C; W represented A or T), where the barcode was an eight-base sequence unique to each sample. The 20-μL PCR reaction mixture was composed of 4 μL of 5× FastPfu buffer, 2 μL of 2.5 mM dNTPs, 5 μM each of forward and reverse primers, 0.4 μL TransStart Fastpfu DNA Polymerase (TransGen Biotech, Beijing, China), and 10 ng DNA template. The following cycling parameters were used: maintain at 95°C for 2 min, 25 cycles (95°C for 30 s, 55°C for 30 s, and 72°C for 30 s), and a final extension at 72°C for 5 min. Triplicate reaction mixtures were pooled per sample, purified using an AxyPrep DNA gel extraction kit (Axygen, Union City, CA, United States) and quantified using a QuantiFluor-ST Fluorescence quantitative system (Promega, Madison, WI, United States). Amplicons from different samples were sent out for pyrosequencing on an Illumina MiSeq platform at Shanghai Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). All sequences have been deposited in the GenBank Short Read Archive (SRP093459).

The sequences in the V4-V5 region of 16S rRNA were selected, and pair-end sequencing was performed in accordance with the requirements of Illumina Miseq high-throughput sequencing. After the target region and fusion primers were designed, two-step polymerase chain reaction (PCR) amplification was performed. The PCR product was recovered by using the AxyPrepDNA gel recovery kit (Axygen Scientific Inc., Silicon Valley, United States). Real-time fluorescence quantification was performed using an FTC-3000TM real-time PCR system (Funglyn, Shanghai, China). The PCR products from different samples were indexed and mixed at equal ratios, to complete the construction of an Miseq library. Then used for high-throughput sequencing and bioinformatics analysis.

A method of 16S rRNA sequencing in colon microbes was used as previously described (16 (link)). In brief, genomic DNA quality control, design and synthesis of primer splices, polymerase chain reaction (PCR) amplification, PCR product purification, quantification, and homogenization, and MiSeq high-throughput sequencing (Illumina, USA) were performed.

The transcribed cDNA obtained from the natural bacterial community was fragmented (~500 bp) with a Covaris focused‐ultrasonicator. The concentration of the obtained cDNA fragments was measured with a QUBIT spectrophotometer (ThermoFisher) following the manufacturer's protocol. The library preparation was performed with a KAPA Hyper kit (KAPA Biosystems). Briefly, 10 ng of cDNA were end repaired and A‐tailed. Subsequently, the genetic material was ligated and purified using a bead‐based cleanup method. The obtained libraries were PCR amplified with following conditions: 98 °C for 45 s, 12 cycles of denaturation at 98 °C for 15 s, annealing at 60 °C for 30 s and extension at 72 °C for 30 s, followed by a final extension at 72 °C for 1 min and hold at 4 °C. The PCR products were further purified using a bead‐based cleanup method. The libraries concentrations were measured by qPCR (KAPA Hyper kit) following the manufacturer's recommendations, and 2.5 nM cDNA of each sample was sequenced using an Illumina MiSeq high throughput sequencing (2 × 250 paired‐end platform) at JAMSTEC, Japan. The sequence data generated are publically available in the DDBJ sequence read archive (DRA) under the accession number DRA008144 for sample t0, DRA008145 for sample t5, DRA008146 for sample t40 and DRA008147 for sample t60.

The process of 16S rRNA sequencing for the colon microbe was performed following a previous study [55 (link)]: genomic DNA quality control, design, and synthesis of the primer splice, PCR amplification and purification of PCR products, quantification and homogenization of PCR products, and MiSeq high-throughput sequencing (Illumina, San Diego, CA, USA).