**2.5 Sequencing analysis**

To get operational sequences, all of the raw sequences obtained from Illumina Miseq were first filtered for quality control. The sequences were checked for quality and analyzed using the software Quantitative Insights into Microbial Ecology (QIIME, v1.8.0) [15]. FLASH [16] was used to combine the paired-end readings from the DNA fragments. Read trimming and identification of V3-V4 sequences were performed on the sequencing data, and a group of sequences with 97% identity was identified as an operational taxonomic unit (OTU). To cluster operational taxonomic units, the UCLUST [17] clustering approach was applied. At a cutoff of 97%, the determined OTUs were allocated to different

*Microbial Diversity and Community Dynamics in the Intestines of Broiler Chicken Raised… DOI: http://dx.doi.org/10.5772/intechopen.103815*

taxonomic levels (phylum, class, genus, and families). Furthermore, the Shannon and Simpson diversity indices, abundance-based coverage estimators (ACE), Chao 1 richness, and coverage percentage were all calculated by Good's method. Also, clustered OTUs were used to construct the rarefaction curves.

#### **2.6 Data analyses and bioinformatics**

The QIIME and R packages were used for bioinformatics and statistical studies (v3.1.1). To determine the relative abundance and diversity of the sequences, the alpha-diversity indices (ACE, Chaol, Shannon, and Simpson index) were calculated. To examine whether taxonomic categories were significantly different across groups of samples based on intestine segments and age period, Metastats and R package (v3.1.1) [18] were used. At p 0.05, the differences were considered significant. A Benjamini–Hochberg false discovery rate correction (Function "p. adjust" in the stats package of R (v3.1.1)) was used to alter the obtained p-value.
