Truseq dna pcr free sample prep kit

Fresh feces were collected, immediately frozen, stored at −80 °C, and subjected to 16 S rRNA sequencing. Microbial genomic DNA extraction was performed with a DNeasyPowerSoil kit (Qiagen, CA, USA). PCR amplicons were purified with a PCR purification kit (Thermo Fisher Scientific, MA, USA) and then quantified, pooled and sequenced with the TruSeqTM DNA PCR-Free Sample Prep kit (Illumina, CA, USA) with PE150 strategy using the standard protocol. The resulting bacterial sequence fragments were sequenced by the Illumina HiSeq X-ten platform using the PE300 mode within a NovaSeq sequencing system. High-quality reads were assigned to respective samples, aligned, and clustered into operational taxonomical units (OTUs) with >97% sequence similarity that simultaneously performs chimaera filtering and OTU clustering. The bacterial sequences were assigned taxonomically based on the SILVA and NCBI databases. Alpha and beta diversity metrics of the samples were calculated using the R package Vegan. Alpha-diversity metrics, including Chao1 and Shannon indices, were used to assess microbial community structures and were computed using R software. Non-metric multidimensional scaling analysis (NMDS) and the principal coordinate analysis (PCoA), based on the unweighted UniFrac distance matrices, were visualized by R software to analyze communities and phylogenesis for the beta diversity.