Fecal microbiota composition analysis in CDKL5 deficiency disorder mouse models

In this study, male CDKL5-/y (CDKL5 KO) mice and their male CDKL5+/y WT littermates were investigated. The composition of the fecal microbiota of CDKL5 KO and WT mice was analysed at different ages. Fresh feces were collected longitudinally from the same subjects at (postnatal day) P25, P32 and P70. We conducted our analysis on two different CDKL5 null mouse lines born and raised in two distinct animal facilities: the vivarium at the National Research Council in Pisa, Italy (Ref. Amendola et al., 2014 PMID: 24838000), and the vivarium at the Max Planck Institute for Molecular Genetics in Berlin, Germany. Bacterial DNA was extracted using the QIAamp Powerfecal DNA kit (Qiagen, Cat. no. 12830-50), and its concentration was quantified by the Nanodrop 2000 C Spectrophotometer (ThermoFisher Scientific). The 16S rRNA sequencing and analysis was performed by a service offered by Institute of Applied Genomics (IGA, Udine, Italy). Library preparation and sequencing. Libraries were prepared by following Illumina 16S Metagenomic Sequencing Library Preparation protocol in two amplification steps: an initial 35 cycle PCR amplification using locus-specific PCR primers and a subsequent amplification that integrates relevant flow-cell binding domains and unique indices (NexteraXT Index Kit, Illumina, FC‐131‐ 1001/FC‐131‐1002). Primer sequences used for amplification: 16S F (341F) 5’- CCTACGGGNGGCWGCAG -3’ 16S R (805R) 5’- GGACTACHVGGGTATCTAATCC -3’ Libraries were sequenced on a MiSeq instrument (Illumina) in paired end 300-bp mode read length. We provide the raw data obtained through the 16S rRNA-sequencing experiment. This dataset contains the following files: 242 files \"fastq.gz\": Files containing the raw sequencing data (FASTQ files) of the 16S rRNA gene profiling carried out on DNA extracted from mouse fecal samples collected longitudinally (at postnatal days (P)-25, P32 and P70) in mice originated from two vivaria: Pisa vivarium (A001) and Berlin vivarium (A002). As sequencing configuration was performed paired-end, each fragment has two raw reads with the first read in forward orientation (R1), and the second read in reverse-complement orientation (R2). Corresponding reads from the R1 and R2 files are collectively a single paired-end read. Adapter sequences masked with MiSeq Reporter, no quality clipping was provided. File Naming Convention (Example): ID2381-2-P25-WT2-A001-B01_S18_L001_R1_001.fastq.gz Explanation of the symbol in the file name: ID2381 = Sequencing ID 2 = Serial number identifying each sequenced fragment (each fragment has two files: R1 & R2) P25 = Postnatal day at collection (P25, P32, or P70- in this case P25) WT2 = Genotype and individual mouse ID (WT = wild-type, KO = knockout, followed by mouse ID number) A001 = Vivarium ID (A001 = Pisa, A002 = Berlin) B01 = Sample batch identifier S18_L001 = Sequencing-specific information R1 = Read direction (R1 = forward, R2 = reverse)本研究针对雄性CDKL5-/y（CDKL5敲除，CDKL5 KO）小鼠及其雄性CDKL5+/y野生型（Wild-type, WT）同窝对照小鼠展开研究。 本研究对不同日龄的CDKL5敲除（KO）与野生型（WT）小鼠的粪便菌群组成进行分析。研究人员对同一批受试小鼠在出生后第25天（P25）、第32天（P32）及第70天（P70）进行纵向新鲜粪便样本采集。 本次分析的受试对象为两个独立的CDKL5全敲除小鼠品系，这两个品系分别在两处不同的动物饲养设施中繁育饲养：一处为意大利比萨国家研究委员会动物饲养房（引用文献：Amendola等，2014，PMID：24838000），另一处为德国柏林马克斯·普朗克分子遗传学研究所动物饲养房。 采用QIAamp Powerfecal DNA提取试剂盒（Qiagen，货号：12830-50）提取细菌基因组DNA，使用Nanodrop 2000 C紫外分光光度计（ThermoFisher Scientific）对DNA浓度进行定量检测。 16S核糖体RNA（16S ribosomal RNA, 16S rRNA）测序及分析工作由意大利乌迪内应用基因组学研究所（IGA）提供的测序服务完成。 文库构建与测序：严格遵循Illumina 16S宏基因组测序文库构建流程进行文库制备，该流程包含两轮扩增步骤：第一轮为35个循环的聚合酶链式反应（Polymerase Chain Reaction, PCR）扩增，使用位点特异性PCR引物；第二轮扩增用于引入测序流动池（flow-cell）结合结构域与独特标签序列，采用NexteraXT索引试剂盒（Illumina，货号：FC‐131‐1001/FC‐131‐1002）。 扩增所用引物序列如下： 16S正向引物（341F）：5’- CCTACGGGNGGCWGCAG -3’ 16S反向引物（805R）：5’- GGACTACHVGGGTATCTAATCC -3’ 采用Illumina MiSeq测序仪进行双端300bp读长模式的测序。 本数据集包含本次16S rRNA测序实验得到的原始测序数据。 本数据集包含以下文件：共计242个fastq.gz格式文件，这些文件存储了针对两处饲养设施（比萨饲养房A001、柏林饲养房A002）的小鼠粪便样本所获得的16S rRNA基因分型测序原始数据（FASTQ格式），上述粪便样本为对受试小鼠在出生后第25、32、70天进行的纵向采集样本。 由于本次测序采用双端测序模式，每个测序片段对应两条原始读段：第一条为正向读段（R1），第二条为反向互补读段（R2）。R1与R2文件中的对应读段共同组成一组双端测序读段。所有测序接头序列已通过MiSeq Reporter软件完成屏蔽，未进行质量修剪操作。 文件命名规范（示例）：ID2381-2-P25-WT2-A001-B01_S18_L001_R1_001.fastq.gz 文件名各字段含义如下： ID2381：测序编号； 2：单测序片段编号（每个测序片段对应R1、R2两个文件）； P25：粪便样本采集时的小鼠出生后天数，可选值为P25、P32、P70，本示例为P25； WT2：小鼠基因型与个体编号，其中WT代表野生型，KO代表敲除型，其后数字为小鼠个体编号； A001：动物饲养房编号，A001对应比萨饲养房，A002对应柏林饲养房； B01：样本批次标识符； S18_L001：测序专属信息； R1：读段方向，R1为正向读段，R2为反向读段