Ion 316 chip kit

Genomic DNA was extracted from the ileum and colon faecal matter using the QIAamp DNA Stool Mini Kit (Qiagen). Colon and ileum DNA were combined and samples were pooled on a group basis for shotgun metagenomic analysis using the Ion Torrent PGM^™ platform. Pooled DNA (100 ng) was fragmented and barcoded using the NEBNext® Fast DNA Fragmentation and Library Prep Set for Ion Torrent^™ (NEB Inc, UK) and IonXpress Barcode Adapters kit (ThermoFisher Scientific) respectively. The quality and quantity of all barcoded DNA libraries were analysed using the High Sensitivity DNA analysis kit (Agilent, UK) on the 2100 Bioanalyzer Instrument and Qubit Fluorometer (ThermoFisher Scientific). Samples were prepared using the Ion PGM^™ Hi-Q^™ View OT2 and Ion PGM^™ Hi-Q^™ View Sequencing kits (ThermoFisher Scientific) and 4 barcoded libraries were combined per Ion 316^™ Chip kit (ThermoFisher Scientific). Data were analysed using MG-RAST and the number of reads per phylum, class, order, family or genera of species of interest were normalized against all bacteria present. Sequencing runs can be accessed using MG-RAST IDs mgl675297, mgl675300, mgl675312, mgl675291, mgl675309, mgl675318, mgl675306, mgl675294, mgl675321, mgl675315, mgl675285, mgl675288, mgl675324, mgl675303.

Emulsion PCR (emPCR) was performed with the Ion PGM Template IA 500 Kit (Thermo Fisher Scientific) and GeneAmp PCR system 9700 (Thermo Fisher Scientific) for transcription analysis of FLA-I genes and with the Ion 520 and 530 Kit-OT2 and OneTouch 2 instrument (Thermo Fisher Scientific) for genotyping of FLA-I and FLA-DRB genes and confirmation of genotyping data in FLA-E/H/K genes. After the emulsion PCR, the beads carrying the single-stranded DNA templates were enriched with the Ion OneTouch Enrichment System (Thermo Fisher Scientific) according to the manufacture’s recommendation. Sequencing was performed using the Ion PGM Hi-Q View Sequencing Kit and Ion 316 Chip Kit (Thermo Fisher Scientific) for transcription analysis and using the Ion S5 Sequencing Kit and Ion 520/530 Chip Kit (Thermo Fisher Scientific) for genotyping of FLA-I and FLA-DRB genes and confirmation of genotyping data in FLA-E/H/K genes.
The raw data processing and base-calling, trimming and output of quality-filter sequence reads that were binned on the basis of the Ion Xpress Barcodes into separate sequence fastq files, were all performed by the Torrent Suite 4.2.1 (Thermo Fisher Scientific) for the transcription analysis and by the Torrent Suite 5.6.0 (Thermo Fisher Scientific) for the genotyping with full processing for shotgun analysis.

cDNA libraries were constructed following the Ion Total RNA-Seq Kit for whole transcriptome protocol (PN 4467091 Rev. C (10/2011)) from Ion Torrent (Life Technologies, Carlsbad, CA, USA). Yield and quality of the libraries were assessed using an Agilent 2100 Bioanalyzer with an Agilent High Sensitivity DNA Kit (Agilent Technologies, Santa Clara, CA, USA) and cDNA were stored at -20°C. The templates were prepared following the Ion OneTouch 200 Template Kit v.2.0 protocol (PN 4478371 Rev. A (06/2012)), and stored at 4°C. Sequencing runs were performed on the Personal Genome Machine following the Ion PGM 200 Sequencing Kit protocol (PN 4474246 Rev. D (06/2012)), using Ion 316 Chip Kit, provided by Life Technologies.