Oral combinatorial antibiotic treatment on the constituency of gut commensal bacteria

16S rRNA metagenomic analysis was performed at Repertoire Genesis (Osaka, Japan). In brief, microbial DNA was extracted from frozen murine fecal samples of SI using Isospin fecal DNA (NIPPON GENE Co., Ltd., Tokyo, Japan). For bioinformatics analysis of 16SrRNA, the V3V4 region of 16S rRNA genes was amplified and sequenced by MiSeq Deep sequencer and MiSeq Reagent Kit v3 (Illumina) following the manufacturer's instructions. The sequence data were preprocessed and analyzed using the Flora Genesis software (Repertoire Genesis Inc.). In brief, the R1 and R2 read pairs were joined and chimera sequences were removed. Operational taxonomic unit (OTU) picking was performed by the open-reference method using the 97% ID prefiltered Greengenes database and uclust. Representative sequences of each OTU were chosen and taxonomy assignment was performed by the ribosomal database project (RDP) classifier using a threshold score of 0.5 or more. The OTUs were grouped if their annotation was the same regardless of the RDP score.

本研究的16S rRNA宏基因组分析工作于日本大阪的Repertoire Genesis公司开展。简言之，研究人员采用日本东京NIPPON GENE株式会社的Isospin粪便DNA提取试剂盒（Isospin fecal DNA），从冷冻保存的SI来源小鼠粪便样本中提取微生物基因组DNA。针对16S rRNA的生物信息学分析，研究人员遵照厂商操作指南，使用Illumina公司的MiSeq深度测序仪（MiSeq Deep sequencer）及MiSeq测序试剂盒v3（MiSeq Reagent Kit v3）对16S rRNA基因的V3-V4可变区进行扩增与测序。后续使用Flora Genesis软件（Repertoire Genesis公司）对测序所得的序列数据进行预处理与分析。简言之，首先将成对的R1与R2测序读段进行拼接，并移除嵌合序列。操作分类单元（Operational Taxonomic Unit，OTU）挑选采用开放参考法，使用经过97%序列一致性预过滤的Greengenes数据库与uclust工具完成。选取每个OTU的代表性序列，并采用核糖体数据库项目分类器（ribosomal database project classifier，RDP classifier）以0.5及以上的阈值得分完成物种分类注释。若不同OTU的物种注释结果一致，则无论其RDP分类得分高低，均将其归为同一类群。