Computational protocol: Transplanted human fecal microbiota enhanced Guillain Barré syndrome autoantibody responses after Campylobacter jejuni infection in C57BL/6 mice

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Protocol publication

[…] In the pilot experiment, DNA was extracted from fecal samples using the QIAamp DNA stool kit (QIAGEN) according to manufacturer’s instructions. DNA concentrations were determined using a NanoDrop ND-1000 spectrophotometer and concentrations normalized. The quantity of Clostridium group 1, Clostridium group 1, Bacteroidetes, and Enterobacteriaceae were measured using an IQTM5 Multicolor Real-Time PCR Detection System. In Experiment 1, DNA was extracted from fecal samples using bead beating and the FastDNA SPIN Kit for Soil (MP Biomedicals, LLC) according to manufacturer’s instructions. The resulting DNA samples were delivered to the Michigan State University Research Technology Support Facility for library preparation and 16S rRNA gene amplicon analysis. In all, 62 samples were submitted for sequencing, including 60 mouse samples, the original fecal slurry used for inoculation of founder mice, and a mock community (HM-782D, BEI) for estimation of sequencing error. The V4 region of the 16S rRNA gene was amplified using dual-indexed primers []. PCR products were normalized using an Invitrogen SequalPrep DNA Normalization plate and the normalized products pooled. After quality control and quantitation, the pool was loaded on a standard MiSeq v2 flow cell and sequenced with a 500 cycle MiSeq v2 reagent kit (paired-end 250 base pair reads). Base calling was performed by Illumina Real-Time Analysis (RTA) v1.18.54 and output of RTA was de-multiplexed and converted to FastQ format files with Illumina Bcl2fastq v1.8.4.16S rRNA gene amplicon analysis was performed using mothur (v. 1.35) and protocols available at http://www.mothur.org/wiki/MiSeq_SOP accessed December, 2015. Alignment was achieved using the Silva 16S ribosomal gene database []. Chimeric sequences and any sequences classified as chloroplast, mitochondria, Archaea, or Eukaryota were removed from the dataset using uchime and the mothur formatted version of the Ribosomal Database Project (RDP) training set version 9, respectively, per the mothur protocol. Sequences were clustered in operational taxonomic units (OTUs) of 97% sequence identity yielding 128 OTUs. Analyses were performed in mothur and PAST 3.07 []. Sequence read data has been made available in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) as documented in “Availability of data and materials.” A full record of the code used to develop the heat map that appears in Fig. , is based on the mothur protocol cited above. An annotated markdown file with the code for the heat map appears in Additional files and . […]

Pipeline specifications

Software tools BCL2FASTQ Conversion Software, mothur, UCHIME
Application 16S rRNA-seq analysis
Organisms Campylobacter jejuni, Homo sapiens, Mus musculus, Bacteroidetes, Firmicutes
Diseases Autoimmune Diseases, Campylobacter Infections, Colitis, Colonic Neoplasms, Gastrointestinal Diseases, Infection, Peripheral Nervous System Diseases, Machado-Joseph Disease, Autoimmune Diseases of the Nervous System, Guillain-Barre Syndrome