Qiaamp r fast dna stool mini kit

Microbial DNA was extracted from segments using the QIAamp R Fast DNA Stool Mini Kit (Qiagen Ltd., Hilden, Germany) according to the manufacturer’s instructions. The V3-V4 regions of the bacteria 16S rRNA gene were generated with universal primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) by PCR amplification [14 (link)]. Shanghai Majorbio Bio-pharm Technology Co. did the sequencing, Ltd. Purified amplicons were pooled in equimolar proportions and sequenced paired-end on the Illumina MiSeq platform (Illumina, San Diego, IL, USA) [15 (link)]. Sequence noise reduction using DADA2, species annotation database as silva138/16s_bacteria, species annotation method as bayes. A Naive Bayes classifier trained on the Greengenes 13 8 99% OTUs was used for taxonomic analysis. The raw sequencing data were processed by a previous study [14 (link)]. R was used to conduct statistical analyses (version 3.3.1). The vegan package was used to compute the Shannon index and richness (mothur-1.30). The data were analyzed on the online platform of Majorbio Cloud Platform (www.majorbio.com (accesed on 21 July 2022). All the raw data were uploaded into the NCBI Sequence Read Archive database with accession number PRJNA876600.

A total of 0.5 g lumen contents and mucosal scraps of ileum, cecum, and colon were used to extract total bacterial genomic DNA using the QIAamp R Fast DNA Stool Mini Kit (QIAGEN Ltd., Hilden, Germany) according to the manufacturer’s protocol. The V3–V4 region of the 16S rRNA gene was amplified using universal primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The PCR reactions were conducted using the following program: denaturation at 95 °C for 3 min, 27 cycles of 95 °C for 30 s, annealing at 55 °C for 30 s, elongation at 72 °C for 45 s, and a final extension at 72 °C for 10 min. The PCR components, the extraction and purification of PCR products were the same with previous study [29 (link)]. Purified PCR products were pooled into equimolar amounts and sequenced on the Illumina MiSeq platform according to the standard protocols by Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China). The raw reads were deposited into the NCBI Sequence Read Archive (SRA) database (Accession Number: PRJNA694358).