Powermag microbiome dna isolation kit

Fecal pellets were collected in collection tubes containing DNA stabilization buffer. Whole metagenome shallow shotgun sequencing (at least 2 million paired-end reads per sample) was performed using the Illumina miSeq or Illumina NextSeq instrument (the instrument used is dependent on the number of samples in a batch, Illumina, San Diego, CA, U.S.A.). Samples were extracted using the Qiagen PowerMag Microbiome DNA Isolation kit (Hilden, Germany) on the King Fisher automated platform (Thermo Scientific, Waltham, MA, U.S.A.). Isolated DNA was quantitated using a fluorescent concentration assay and normalized to prepare for library preparation using the Illumina Nextera XT DNA Library prep recommendations. The runs were spiked with 1% PhiX. Standard processing used 2 × 150 base pair paired-end sequencing with dual 8 base pair indexes. The instrument run took approximately 29 h. Criteria for acceptable results: The final run must have a cluster density of 180–230 K/mm² with >80% of clusters passing the filter, and at least 75% of bases must call at a minimum Phred score of Q30 (99.5%).

At 12 weeks of age, mice (n = 9/genotype) were single housed for 2 weeks and colon content was collected into collection tubes containing DNA stabilization buffer. Whole metagenome shallow shotgun sequencing (at least 2 million paired-end reads/sample) was performed using the Illumina miSeq or Illumina NextSeq instrument (the instrument used is dependent on the number of samples in a batch, Illumina, San Diego, CA). For the analysis, samples were extracted using the Qiagen PowerMag Microbiome DNA Isolation kit (Hilden, Germany) on the King Fisher automated platform (Thermo Scientific, Waltham, MA). Isolated DNA was quantitated using a fluorescent concentration assay and normalized to prepare for library prep using the Illumina Nextera XT DNA Library prep recommendations. Runs were spiked with 1% PhiX. Standard processing used 2 x 150 base pair paired-end sequencing with dual 8 base pair indexes. The instrument run takes ~29 hours. Criteria for acceptable results: final run must have a cluster density of 180-230K/mm² with greater than 80% of clusters passing filter, and at least 75% of bases must call at a minimum Phred score of Q30 (99.5%).