Dna stool kit

Bacterial genomic DNA was isolated from each fecal sample using the DNA Stool Kit (Qiagen Bioinformatics Co., Ltd, Hilden, Germany). Metagenomic shotgun sequencing libraries were constructed and sequenced at Suzhou PANOMIX Biomedical Tech Co., LTD (Suzhou, China). The alpha diversity is assessed using three parameters (Simpson, Chao, Shannon), and the beta diversity is assessed using PCoA. Besides, the LEfSe is executed to identify the significantly different species. Moreover, functional annotation of the KEGG orthologs (KOs) was performed using LEfSe analysis. Subsequently, integrating taxa at phylum, genus, and species levels and SCFAs, the correlation coefficients between the two components as a heatmap were determined using Spearman analyses using the corrplot package in R.

Eight fecal samples were obtained from each of the eight mice in each group at the end of the 8-week intervention period. Bacterial chromatin was extracted using a DNA Stool kit (Qiagen Bioinformatics Co., Hilden, Germany) according to the manufacturer’s protocol. Subsequently, PCR was performed after ligating the adapters, size selection, and tailed random primers to obtain sufficient amplification products for library construction. The libraries were prepared using the NEBNext Ultra DNA Library Prep Kit for Illumina, and their quality was validated with a DNA LabChip 1000 kit on an Agilent 2100 Bioanalyzer (Agilent Technologies, Wokingham, UK). Clusters were generated by bridge amplification within paired-end flow cells using the Illumina TruSeq PE Cluster Kit v3-cBot-HS, according to the manufacturer’s instructions. After cluster generation, paired-end sequencing was performed on an Illumina HiSeq 2000 platform using the TruSeq SBS Kit v3-HS. Unpaired reads were excluded from the clean reads. High-quality sequencing reads were de novo assembled into long contigs or scaffolds, which were used for gene prediction, taxonomic classification, and functional annotation. Detailed process for metagenomic shotgun sequencing was provided in online supplemental additional file 1.

Hh NCI-Frederick isolate 1A (strain 51449) was grown as described previously [14] (link). Seven day old BALB/c mice from Helicobacter-free breeders were fed on two consecutive days with Hh 1A (∼2.5 × 10⁷ CFU) by oral gavage. Hh colonisation was analyzed in caecal contents collected upon sacrifice. DNA was isolated using the DNA Stool kit (QIAGEN) and SYBR Green real-time PCR with Hh-specific primers against the cdtB gene (Fwd: CCG CAA ATT GCA GCA ATA CTT; Rev: TCG TCC AAA ATG CAC AGG TG) was performed in triplicate using the CFX96 detection system (Bio-Rad Laboratories). Results represent arbitrary units normalised to uninfected control samples.