16S rRNA (gene) based barcoded Illumina sequencing of the microbiota in colonic luminal content and fresh faeces of mice during aging
Microbiota composition was determined in the colonic luminal content of 10 mice per group aged 6, 12, 24 and 28 months, resulting in a total of 40 samples. Fresh faeces was collected during the life span of 9 mice at the age of 4, 8, 12, 18, 24 and 28 months, resulting in a total of 42 samples.Illumina adaptor sequences. Amplicons were purified using the ZR-96 DNA Clean and Concentrator-5 Kit (ZYMO Research, Irvine, CA, USA). To quantify DNA content of the resulting amplicon, the Qubit Fluorometric Quantitation device (ThermoFisher Scientific, Waltham, MA, USA) was used. Besides, the amplicons were run on a 2%-agarose gel to confirm amplicon size and amount. Purified PCR products were used for the second PCR in combination with sample-specific barcoded primers (Nextera XT index kit, Illumina, San Diego, CA, USA). Purified PCR products with an amplicon length of 550 base pairs (bp) (including adaptors) were submitted for sequencing in the paired-end (2x) modus (300 bp) on the MiSeq platform (Illumina, San Diego, CA, USA) at the BaseClear service laboratory (BaseClear BV, Leiden, The Netherlands). FASTQ files were generated and de-multiplexed based on sample-specific barcodes using the Casava pipeline (version 1.8.3, Illumina, San Diego, CA, USA). Sequence reads of low quality (only "passing filter" reads were selected) or containing the adapters or PhiX control signals were removed and the FASTQC pipeline (version 0.10.0, (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) was applied for the initial quality control.