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)