Geneamp 2720

Total DNA was extracted according to the instructions of the Power Water^® DNA Isolation Kit (MO BIO laboratories, Inc., Carlsbad, CA, USA). The concentration and purity of the DNA extracts were measured with a NanoDrop2000 (Thermo Fisher Scientific, Waltham, MA, USA), and the quality of the DNA extracts was determined by 1% agarose gel electrophoresis. PCR amplification of the V3–V4 hypervariable regions of the 16S rRNA gene was performed using 338F(5’-ACTCCTACGGGAGGCAGCAG-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’) primers [16 (link)]. The PCR protocol was as follows: initial denaturation at 98 °C for 2 min, 27 cycles of (denaturation at 98 °C for 15 s, annealing at 55 °C for 30 s, extension at 72 °C for 30 s), and finally extension at 72 °C for 5 min. Each sample involved three technical replicates for 16S rRNA gene amplification, which was carried out in a GeneAmp 2720 thermocycler (Applied Biosystems, Foster City, CA, USA). Indexing PCR was performed in a 25 μL reaction system containing 5 μL reaction buffer (5×), 5 μL GC buffer (5×), 2 μL 2.5 mM dNTP, 1 μL Forward primer (10 μM), 1 μL Reverse primer (10 μM), 0.25 μL Q5^® High-Fidelity DNA Polymerase, 2 μL template DNA, and 8.75 μL ddH2O.

PCR amplification was performed in a total reaction volume of 20 μL containing 10 μL of Platinum^® Multiplex Master Mix (Thermo Fisher Scientific, MA, USA), 2.4 μL GC Enhancer, 5.6 μL of the eight SNP-STRs primer mixture (Table 1) and 1 ng of DNA template. Thermal cycling was performed on GeneAmp 2720 (Thermo Fisher Scientific, MA, USA) under the following conditions: 95°C for 2 min; 30 cycles of 95°C for 30 s, 60°C for 90 s, 72°C for 35 s, and a final extension hold at 72°C for 10 min.
PCR products were electrophoresed on ABI 3130 Genetic Analyser (Thermo Fisher Scientific, MA, USA) following manufacturer’s protocols. Samples were prepared as a mixture of 0.3 μL GeneScan™ 500 LIZ^® size standard (Thermo Fisher Scientific, MA, USA) with 8.7 μL Hi-Di™ Formamide (Thermo Fisher Scientific, MA, USA) and 1 μL PCR products. Samples were analyzed using GeneMapper ID v3.2 software (Thermo Fisher Scientific, MA, USA) after data collection.

Genomic DNA was extracted using the Chelex-100 method [17 (link)], and quantified by the Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, MA, USA). The multiplex PCR amplification of the 32 autosomal InDels were conducted according to the PCR protocol described in our previous research [11 (link)] on GeneAmp 2720 (Thermo Fisher Scientific, MA, USA). The PCR products were subsequently separated and detected via the ABI 3130 Genetic Analyzer (Thermo Fisher Scientific, MA, USA), and were analyzed by the GeneMapper ID v3.2 software (Thermo Fisher Scientific, MA, USA).