Miseq reagent kits v3

Total DNA was extracted from stool samples of 20 mice using the QIAamp^®DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocols. DNA extracts were determined by agarose gel electrophoresis (1% w/v agarose) and quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific).
The V3-V4 region of the 16S rRNA gene was amplified by PCR using a 30 μl mixture containing 0.5 μl of DMSO, 1.0 μl of 319F (10 mM), 1.0 μl of 806R (10 mM), 5.0 μl of the DNA sample, 7.5 μl of ddH₂O, and 15.0 μl of Phusion High-Fidelity PCR Master Mix with HF Buffer (NEB) (He et al., 2016 (link)). The reactions were hot-started at 98°C for 30 s, followed by 30 cycles of 98°C for 15 s, 58°C for 15 s, and 72°C for 15 s, with a final extension step at 72°C for 1 min. Subsequently, the amplicons were purified according to standard procedures, quantified, pooled and sequenced with the MiSeq Reagent Kits v3 (600 cycles, Illumina) according to the manufacturer’s instructions with 20% OhiX (Illumina). The sequencing reaction was conducted by Hangzhou Guhe Information and Technology Co., Ltd., Zhejiang, China.

The PA gene was amplified from viral RNA using the SuperScript III One‐step RT‐PCR system with Platinum Taq (Thermo Fisher Scientific) and universal primers.17 A DNA library was prepared from RT‐PCR products using a QIAseq FX DNA Library Kit (Qiagen), followed by purification by Agencourt AMPure XP (Beckman Coulter). The library was sequenced with MiSeq Reagent Kits v3 and the MiSeq system (Illumina). Sequence reads were aligned to reference sequences using CLC Genomics Workbench 11 (Qiagen). A minor allele frequency threshold of 10% was used for the detection of SNPs. All sequences are available from the EpiFlu database of the Global Initiative on Sharing All Influenza Data (GISAID) (Table 3).

All solutions containing the extracted DNA were diluted to a 5 ng/µL concentration and then 20 ng (corresponding to 4 µL) of each sample were used for sequencing by next-generation sequencing (NGS). This was carried out in the molecular biology laboratory of the University of Catania on an Illumina MiSeq platform according to the manufacturer’s instructions provided in the AmpliSeq^TM Cancer HotSpot Panel v2 for Illumina^® (Ref. 20019161, Illumina, Inc., 92122, San Diego, CA, USA); in the end, we were able to sequence 2800 COSMIC mutations from 50 oncogenes and tumor suppressor genes. Indexes were provided with AmpliSeq^TM CD Indexes, Set A for Illumina^® (96 Indexes, 96 Samples) (Ref. 20019105, Illumina, Inc., 92122, San Diego, CA, USA). Denature and dilute libraries were obtained following the “Denature and Dilute Libraries Guide” protocol provided by Illumina^® (Document # 15039740 v10), choosing as loading concentration 9 pM. Finally, sequencing was performed using the MiSeq Reagent Kits v3 (Ref. 15043895, Illumina, Inc., 92122, San Diego, CA, USA). The creation of the sample sheet was accomplished by using the Local Run Manager v3 software and following the instructions in the Local Run Manager v3 Software Guide provided by Illumina.