title: yolksac sample
design: DNA Extraction and High-Throughput SequencingBacterial DNA was extracted from ileal digesta with a QIAamp DNA Stool Mini Kit (Qiagen Inc., V alencia, CA) according to the manufacturers protocol. The concentrations of DNA extracts were measured on a NanoDrop 2000 spectrophotometer (Thermo Scientific, MA, USA). The V4 region of the bacterial 16S rRNA gene was amplified with the barcoded primer pair 515F/806R (515F: 5-GTG CCA GCM GCC GCG GTA A-3, 806R: 5-GGA CTA CHV GGG TWT CTA AT-3) according to previously described methods (Wang et al., 2017). After amplification, PCR products run on a 2% agarose gel and were purified using a QIAquick Gel Extraction Kit (Qiagen, Germany). Pyrosequencing of 16S rDNA was performed on an Illumina HiSeq2500 PE250 platform (Illumina, San Diego, USA) at Novogene Bioinformatics Technology Co. Ltd. (Beijing, China).Sequence Processing and Bioinformatics AnalysisRaw tags were generated by merging paired-end reads using FLASH software (v1.2.7) (Magoc and Salzberg, 2011). Highquality clean tags were obtained by QIIME (v1.7.0) analysis (Caporaso et al., 2010), and chimera sequences were removed to obtain effective tags by using the UCHIME algorithm (Edgar et al., 2011). Sequences were analyzed by UPARSE software (v7.0.1001) and clustered into operational taxonomic units (OTUs) at a similarity level of 97% (Edgar, 2013). Each OTU was annotated with the Greengenes database (DeSantis et al., 2006). Rarefaction curve and Venn diagram were created using R software (v2.15.3). Analysis of microbial alpha diversity was conducted using QIIME software (Caporaso et al., 2010) with Python scripts. Beta diversity was evaluated by principal component analysis (PCA) to show the differences of bacterial community structures, and the significance of separation was tested via ANOSIM using R (v2.15.3). PICRUSt analysis was used to predict the functional potential of bacteria communities (Langille et al., 2013). OTUs were normalized by copy number, and metagenome prediction was further categorized into Kyoto Encyclopedia of Genes and Genomes (KEGG) at levels 2 and 3 (Kanehisa et al., 2094).