Illumina PE250 High Throughput Sequencing

The total DNA of the feces were extracted by the DNA extraction kit. The DNA extraction was subjected to PCR amplification of the 16S rRNA gene V3-V4 region and high-throughput sequencing of Illumina PE250 by Shanghai Lingen Biomedical Technology Co., Ltd. (Shanghai, China). Specific primers with barcode were synthesized for the 16S rRNA gene. The PCR amplification was carried out using TransStart Fastpfu DNA polymerase kit. The program as follow: one cycle of 95°C for 300 s followed by 27 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 45 s, together with three replicates per sample. The PE reads were first spliced according to the overlap relationship. Meantime, the quality of the sequence was quality-controlled and filtered, and the OTU cluster analysis and species taxonomic analysis were performed after distinguishing the samples. Based on the above analysis, alpha diversity and beta diversity indices were calculated with QIIME (Version 1.7.0). To mine the microbial diversity difference among samples, significance test was performed by linear discriminate analysis effect size (LEfSe). To predict the functional profiles of the microbial communities, phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) analysis was performed, estimating the functions based on 16S rRNA sequencing data, Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, and KEGG Orthology (KO) databases.