Ion plus fragment library kit 48 rxns library construction kit

The genomic DNA was extracted according to the method of Chen et al. [20 (link)]. The PCR amplification was performed on the V3-V4 hypervariable regions of the bacterial 16S rDNA genes, which used the forward primer 341F (5′-CCTAYGGGRBGCASCAG-3′) and the reverse primer 806R (5′-GGACTACNNGGGTATCTAAT-3′). PCR products were accumulated using the QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany). The 16S rDNA amplicon sequencing was performed on the Illumina NovaSeq PE250 platform (Novogene Bioinformatics Technology Co., Ltd., Beijing, China). The Ion Plus Fragment Library Kit 48 rxns library construction kit of Thermofisher Company (Waltham, MA, USA) was used to construct the library. After the library was qualified by Qubit quantification and a library test, the Ion S5TMXL of Thermofisher was used for computer sequencing. Clean reads of all samples were clustered by Uparse software (Uparse v7.0.1001, Robert C Edgar, Tiburon, CA, USA,) at a 97.0% similarity level to obtain OTUs. Alpha diversity analysis was performed using ACE, Chao 1, rarefaction curves, and Shannon indexes. The weighted unifrac algorithm was used for the analysis of PCoA. The analysis of LefSe was performed with a threshold >4.0.

Approximately 0.4 g (approximately 4 pieces) of fresh feces was taken from the GOS-H group, placed in a sterile centrifuge tube, rapidly frozen with liquid nitrogen, and stored at −80 °C for future use. Genomic DNA was extracted and amplified by PCR using a DNA extraction kit. The universal primers 515F and 806R were used to amplify the V3-V4 region of the bacterial 16S rRNA gene (Table S2). The 16S rRNA gene sequencing library of bacteria was prepared by using Thermo Fisher’s Ion Plus Fragment Library Kit 48 RXNS Library construction kit. After the constructed library was qualified by with a Qubit and library detection was performed, an Ion S5TMXL Thermo Fisher instrument was used for on-machine sequencing.
Using UPARSE software, clean reads were clustered into operational taxonomic units (OTUs) according to the consistency of paired nucleotide sequences with a threshold of 97%, and the sequences with the highest frequency in OTUs were considered representative OUT sequences. Based on the above analysis, alpha diversity analysis and beta diversity analysis were performed. In addition, t tests and linear discriminant analysis of effect size (LefSe) were used to identify significant differences in species among the groups to better understand the differences in bacterial communities between the groups.