Miseq 2000 sequencer

Library preparation was performed using the 16S Metagenomics Sequencing Library Preparation kit according to the manufacturer’s protocol (Illumina, Inc, San Diego, CA, USA). Quality control of the sample library and quantification of the DNA library templates was performed. Quantification of DNA was done using Qubit^® fluorescent dye method. The library size distribution was checked using a High Sensitivity DNA chip. Thereafter, the indexed libraries were normalized, pooled, and loaded onto an Illumina MiSeq reagent cartridge using MiSeq reagent kit v3 and 600 cycles. The paired end 2 × 300 bp sequencing was run on an Illumina MiSeq 2000 sequencer at 0.2× coverage at the Biotechnology Platform, Agricultural Research Council, Onderstepoort, South Africa.

A fresh fecal sample was collected by participants at the same visit of neuropsychological testing for MCI diagnosis and mailed to Germark Biotechnology (Taichung, Taiwan). Bacterial DNA was extracted using QIAamp Fast DNA Stool Mini Kit (Qiagen, MD, United States). The amount and quality were then determined by NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, United States). Extracted DNA was stored at –80°C before 16S rRNA sequencing. V3-V4 regions of bacterial 16S rRNA gene, which are hypervariable, were amplified by PCR using bar-coded primers 341F (forward primer; 5’-CCTACGGGNGGCWGCAG-3′) and 805R (reverse primer; 5’-GACTACHVGGGTATCTAATCC-3′) (Herlemann et al., 2011 (link)). Sequencing and library construction of amplicon DNA samples were entrusted to Germark Biotechnology (Taichung, Taiwan). Then, the 2 × 300 bp paired-end amplicon library with an insert size of 465 bp for each sample was prepared using the TruSeq Nano DNA Library Preparation kit (Illumina Inc., San Diego, CA, United States). At last, high-throughput sequencing was performed on an Illumina MiSeq 2000 sequencer with MiSeq Reagent Kit v3 (Illumina).