16SrRNA reveal how green tea polyphenols against the Eimeria tenella infection in Wuliangshan black-bone chickens
The caecal contents was collected from group5 (not treated ).DNA of caecal contents was extracted using the CTAB according to manufacturers instructions. PCR amplification was performed using V3-V4 primers in a total volume of 25 L reaction mixture containing 25 ng of template DNA. The PCR products were purified by using AMPure XT beads (Beckman Coulter Genomics, Danvers, MA, USA) and quantified by Qubit (Invitrogen, USA). The abundance and quantity of the amplicon library were assessed using Agilent 2100 Bioanalyzer (Agilent, USA) and the Library Quantification Kit for Illumina (Kapa Biosciences, Woburn, MA, USA), respectively. The libraries were sequenced on NovaSeq PE250 platform. Paired-end reads was assigned to samples based on their unique barcode and truncated by cutting off the barcode and primer sequence. Paired-end reads were merged using FLASH software. Qualities filtering on the raw reads were performed by using fqtrim (v0.94). Chimeric sequences were filtered using Vsearch software (v2.3.4). After dereplication using DADA2, we obtained feature table and feature sequence. Alpha diversity and beta diversity were calculated by normalized to the same sequences randomly. Then according to SILVA classifier, feature abundance was normalized using relative abundance of each sample. Alpha diversity was applied for analyzing complexity of species diversity through 4 indices, including Chao1, Shannon, Simpson, and all these indices in our samples were calculated with QIIME2. Beta diversity was calculated by QIIME2, the graphs were drawn by R package. Blast was used for sequence alignment, and the feature sequences were annotated with SILVA and NT-16S database using representative sequence.