Premix ex taq hot start version

Genomic DNA was bisulfite-converted using the EZ DNA Methylation-Gold kit (ZYMO, Tustin, CA, USA) according to manufacturer’s instructions. Bisulfite-converted DNA was amplified by PCR using Premix Ex Taq™ Hot Start Version (TaKaRa, Dalian, China) according to manufacturer’s instructions. Primers for BS-PCR were designed using the online MethPrimer program (http://www.urogene.org/cgibin/methprimer2/Meth Primer.cgi) (Supplementary Table S1). PCR products were purified and cloned into the pMD19-T vector (TaKaRa, Dalian, China), and six clones, selected from each sample, were sent to Sangon Biotech Co. Ltd. (Shanghai, China) for sequencing. The sequencing results were analyzed using the online Quma software (http://quma.cdb.riken.jp/) for individual CG sites and DNA methylation levels.

To screen the potential inner primers, 40 cycles of PCR were conducted twice using selected water samples (N = 6) from Sample Set 1. The six samples were collected between April and September, which includes both epidemic and non-epidemic seasons, and RVA concentrations were in the range of 4.1–5.5 log₁₀ copies/L. Complementary DNA (cDNA) was obtained through RT using the PrimeScript RT Master Mix (Perfect Real Time, Takara Bio Inc. Kusatsu, Japan). Briefly, 100 μl of reaction mixture contained 20 μl of 5 × PrimeScript RT Master Mix, 50 μl of viral RNA extracts, and 30 μl of deionized distilled water. RT was conducted at 37°C for 15 min and 42°C for 5 min, and enzyme inactivation was conducted at 85°C for 5 s.
PCR was performed in a 25-μl reaction mixture containing 12.5 μl of the Premix Ex Taq Hot Start Version (Takara Bio Inc.), 400 nM each of forward and reverse primers, and 3 μl of cDNA. The PCR conditions were: 40 cycles at 98°C for 10 s, 55°C for 30 s, and 72°C for 30 s. PCR products were purified using the QIAquick PCR Purification Kit (Qiagen). Then, 3 μl of the purified PCR products was subjected to a second PCR cycle under the same amplification conditions with the same inner primer sets. The PCR products were visualized using 1.5% agarose gel electrophoresis.

Cecal digesta were collected, and total DNA was extracted using a QIAamp DNA stool MiniKit (Qiagen, Hilden, Germany). PCR reactions targeting the V3–V4 regions of the 16S rDNA genes (primers: 319F, 5′-ACTCCTACGGGAGGCAGCAG-3′; 806R, 5′-GGACTACHVGGGTWTCTAAT-3′) were performed using the Premix Ex Taq Hot Start Version (Takara, Dalian, China) on a gradient PCR instrument. The amplicons were then purified using an AxyPrep DNA gel extraction kit (Axygen Bioscience, Union City, USA). The sequencing reactions were subsequently assessed using an Illumina Miseq PE250 platform (Shanghai Majorbio Bio-pharm Biotechnology Co., China). Raw reads were merged using FLASH (v1.2.11) and then analyzed using QIIME (v1.9.0). The sequences were binned into operational taxonomic units (OTUs) on the basis of 97% identity. Taxonomy analyses were performed using the Ramer–Douglas–Peucker (RDP) algorithm of the Greengenes database. Alpha diversity indices, such as Chao1, Shannon, and Simpson diversity indices, were chosen to estimate the species richness using the Mothur program. Principal coordinate analysis (PCoA) was used to represent the relationships between the samples on the basis of calculations of UniFrac metrics. Heat map and the hierarchical clustering tree at the genus level was calculated based on a UniFrac metrics dissimilarity matrix using Vegan 2.0 packages in R (version 3.1.2) software.

To validate predicted AS events, the selected ES, RI, A5SS, and A3SS events were validated by polymerase chain reaction (PCR) using a set of primers (Supplementary Table 1) that were designed based on each AS event. For each sample, 1.5 μg total RNA was reverse transcribed into first-strand cDNA using a PrimeScript RT reagent kit (TaKaRa). PCR was performed in a 25-μl reaction system using Premix Ex Taq™ Hot Start Version (TaKaRa), and the procedure was as follows: initial denaturation at 95°C for 1 min; 95°C for 10 s, 55°C for 30 s, and 72°C for 1 min for 35 cycles; and a final extension at 72°C for 5 min. The PCR products were visualized by 1.5% agarose gel electrophoresis.

Bacterial DNA was extracted from fecal samples, amplified, and sequenced [30 (link)]. Microbial community analysis based on 16S rRNA gene sequences was conducted using the QIIME software package version 1.8.0. In brief, the hypervariable regions (V1–V2) of 16S rRNA gene sequences were amplified using multiplex identifier adaptor primers (454 Life Sciences). The barcode primer set and approximately 10 ng of template DNA were added to a 50 μL PCR mix (Premix Ex Taq Hot Start Version, TaKaRa) for amplification under the following conditions: 94°C for 3 min, followed by 20 cycles at 94°C for 15 s, 55°C for 45 s, and 72°C for 1 min, and a final extension at 72°C for 8 min. Three independent PCR replicates for each sample were pooled, quantified using a spectrophotometer (NanoDrop ND-2000, Thermo Fisher Scientific), and combined in equimolar ratios. Pooled DNA was sequenced using a 454 GS FLX Titanium pyrosequencer (Roche 454 Life Sciences).