Fast dna spin extraction kit

Fecal samples served as the substrate for the total bacterial genomic DNA using the Fast DNA SPIN extraction kits (MP Biomedicals, Santa Ana, CA, USA). DNA molecular size and quantification were performed using a 0.8% agarose gel electrophoresis and the NanoDrop NC-2000 spectrophotometer, respectively. The V3–V4 region of bacterial 16S rRNA genes was then amplified using PCR with the forward primer 338F (5'-ACTCCTACGGGAGGCAGCA-3') and the reverse primer 806R (5'-GGACTACHVGGGTWTCTAAT-3'). PCR amplicons were purified with Agencourt AMPure Beads (Beckman Coulter, Indianapolis, IN) and quantified using the PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). Finally, the MiSeq Reagent kit v3 (Shanghai Personal Biotechnology Co., Ltd, Shanghai, China) was used to carry out the sequencing on the Illumina MiSeq platform.

Spontaneous or induced sputum was collected after having the subjects swish sterile water in their mouths and placed in DNA-free Petri dishes. Sputum plugs, which contained the most viscous material, were then picked up and isolated from saliva. All samples were weighed and frozen at −80°C until sample processing. Negative control samples consisted of unused sterile water and DNA contamination of reagents and equipment was evaluated using extraction controls, which were processed and analyzed alongside the experimental samples.
After thawing of the sputum samples, total bacterial genomic DNA samples were extracted using the Fast DNA SPIN extraction kits (MP Biomedicals, Santa Ana, CA, USA), following the instructions of the manufacturer. The quantity and quality of extracted DNA were measured using a NanoDrop ND-1000 spectrophotometer and agarose gel electrophoresis, respectively.

Total genomic DNA extraction was carried out using the Fast DNA SPIN extraction kits (MP Biomedicals, Santa Ana, CA, United States) according to the manufacturer’s instructions. For bacteria, the V3–V4 hypervariable region of the 16S rRNA gene was amplified by PCR with the universal primers of the forward 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and the reverse 806R (5′-GGACTACHVGGGTWTCTAAT-3′). For fungi, the ITS1 was amplified by PCR with the primers of ITS5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′) and ITS1 (5′- GCTGCGTTCTTCATCGATGC-3′). After the individual quantification, amplicons were pooled in equal quantities and subjected to high-throughput sequencing with a MiSeq Reagent Kit V3 (Personal, Shanghai, China) for pair-end 2 × 250 bp sequencing.
The sequences were analyzed with QIIME2 (2019.4), and the quality control of the sequences by using DADA2 (Callahan et al., 2016 (link)). Specifically, the default parameters of DADA2 were used for filtering and denoising. Secondly, the sequences were automatically calculated and spliced according to the clip length. Thirdly, the sequences with chimerism >8 were removed. Finally, the sequences with low abundance (reads ≤1) were removed. Each de-weighted sequence resulting from quality control using DADA2 is called ASV.