Geneamp 9700

cDNAs were synthesized using a SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific) and used for NGS library preparation. The libraries were manually constructed using our custom thyroid fusion panel, ThyChase. The amplicon library was prepared with the Ion Plus Fragment Library Kit (Life Technologies, Waltham, MA, USA) and the Ion Xpress Barcode Adapters Kit (Life Technologies) according to the manufacturer's instructions. In detail, 10 μL of cDNA was amplified in reaction mixtures of 59 μL containing 45 μL of Platinum PCR SuperMix High Fidelity and 4 μL of ThyChase panel. Polymerase chain reaction (PCR) was performed with a GeneAmp 9700 thermal cycler (Thermo Fisher Scientific) under the conditions of 95°C for 2 min followed by 35 cycles of 95°C for 15 s, 58°C for 15 s, 68°C for 10 s, and a final hold at 4°C. Libraries were purified using 106 μL of AMPure XP Reagent (Beckman Coulter, Miami, FL, USA) on a magnetic stand (Thermo Fisher Scientific) and eluted with 25 μL of low tris-EDTA buffer. Then, adapter ligation and nick repairing were performed to make barcode sequencing adapters (Ion Xpress Barcode Adapters, Thermo Fisher Scientific). Finally, the libraries were quantified using quantitative PCR (qPCR; Ion Library Quantitation Kit, Thermo Fisher Scientific) on a QuantStudio 12K Flex Real-Time PCR System qPCR machine (Thermo Fisher Scientific).

Nine samples from three different regions, each with three duplicates, had their total DNA extracted using DNA extraction kits in strict accordance with the manufacturer’s instructions. A NanoDrop 2000 UV-Vis spectrophotometer (Thermoscientific, Waltham, MA, USA) was then used to analyze the DNA concentrations. Using the thermal cycling PCR technique, the V3-V4 hypervariable region of the bacterial 16S rRNA gene was amplified (Gene Amp 9700, ABI, Thermo Fisher Scientific Co., Ltd., Waltham, MA, USA). With three duplicates of each sample, all tests were conducted in accordance with formal experimental guidelines. PCR products were recovered by gel cutting with the AxyPrepDNA Gel Recovery Kit (AXYGEN, Shanghai, China), eluted with Tris HCl, and identified by 2% agarose gel electrophoresis. PCR products from the same sample were mixed together and detected using this method.

The V3-V4 hypervariable regions of the bacteria 16S rRNA gene were amplified with primers 338F (5’-ACTCCTACGGGAGGCAGCAG-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’) by thermocycler PCR system GeneAmp 9700 (Thermo Scientific). The PCR products were purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and quantified using QuantiFluor™-ST (Promega, Madison, WI, U.S.A.) according to the manufacturer’s protocol. Purified amplicons were pooled in equimolar and paired-end sequenced (2×300 cycle run) on an Illumina MiSeq platform (Illumina, San Diego, CA, U.S.A.) according to the standard protocols outlined by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China).