title: 16S V3-V4 Amplicon seq of human: Diabetic patient 2
design: he DNA was extracted from human stool using InvitrogenTM PureLinkTM Microbiome DNA purification Kit and sent to Novogene Co., Ltd. in Singapore for deep sequencing of the region V4 of the partial 16S rRNA gene. The V4 region of the bacterial 16S rRNA gene was then amplified by PCR using the barcode-encoded primers 515F and 806R. 515F (5- GTGCCAGCMGCCGCGGTAA-3) and 806R (5-GGACTACHVGGGTWTCTAAT-3). All PCR reactions were carried out with Phusion High-Fidelity PCR Master Mix with GC Buffer (New England Biolabs), using 25l Taq PCR mix*2, 1l 10uM Primer F, 1l 10uM Primer R, 2.5l gDNA, 8.0l H2O. An initial denaturation step of 95 C for 5 min was carried out, followed by 34 cycles of denaturation (94 C, 1 min), annealing (57 C, 45 s) and extension (72 C, 1 min), and a final elongation step of 10 min at 72 C cooling for 5 mine at 16 C. Samples with bright main strip between 400-450bp were chosen for further experiments. PCR products was mixed in equinity ratios. Then, mixture PCR products were purified using Qiagen Gel Extraction Kit (Qiagen, Germany). Sequencing libraries were created NEBNext UltraTM DNA Library Prep kit for Illumina following manufacturer's recommendations and index codes were added. Using a Thermo Scientific Qubit@ 2.0 Fluorometer, the library's quality was evaluated. Once prepared, the libraries were sequenced on an Illumina HiSeq. 2500 platform resulted in the recovery of ~250 bp paired-end raw reads . Following that, bioinformatics analysis was carried out.