Qiaxcel dna fast analysis

DNA extractions were processed at the University of Connecticut’s Microbial Analysis, Resources, and Services facility for 16S rRNA gene amplifications and sequencing. Bacterial 16S rRNA genes were amplified using 30 ng of extracted DNA from each of the 192 soil samples. The V4 region was amplified using primers 515F (GTGYCAGCMGCCGCGGTAA) and 806R (GGACTACNVGGGTWTCTAAT) with Illumina adapters and dual indices. Samples were amplified in triplicate using GoTaq (Promega) with the addition of 10 μg BSA (New England BioLabs), and the three independent PCR reactions were pooled prior to sequencing. The PCR reaction was incubated at 95∘C for 3.5 min, then 30 cycles of 30 s at 95.0∘C, 30 s at 50.0∘C and 90 s at 72.0∘C, followed by final extension as 72.0∘C for 10 min. PCR products were pooled for quantification and visualization using the QIAxcel DNA Fast Analysis (Qiagen). PCR products were normalized based on the concentration of DNA then pooled using the QIAgility liquid handling robot. The pooled PCR products were cleaned using the Mag-Bind RxnPure Plus (Omega Bio-tek) according to the manufacturer’s protocol. The cleaned pool was sequenced on the MiSeq platform using v2 2 × 250 chemistry (Illumina, Inc).

DNA was extracted from whole tissue biopsies (proximal ACF and adjacent normal mucosa) using the MoBio PowerMag Soil 96 well kit (MoBio Laboratories) according to the manufacturer’s protocol for the Eppendorf epMotion liquid-handling robot. DNA extracts were quantified using the Quant-iT PicoGreen kit (Thermo Fisher Scientific). The V4 hypervariable region of the bacterial 16S rRNA gene was amplified using 30 ng of extracted DNA as a template and the primer set of 515 F and 806 R with Golay code indices.^{43 (link)} Samples were amplified in triplicate using GoTaq (Promega) with the addition of 10 µg of BSA (New England BioLabs). The PCR reaction was incubated at 95 °C for 3.5 min with 30 cycles of 30 s at 95.0 °C, 30 s at 50.0 °C, and 90 s at 72.0 °C, followed by a final extension at 72.0 °C for 10 min. PCR products were pooled for quantification and visualization using the QIAxcel DNA Fast Analysis (Qiagen). PCR products were normalized based on the concentration of DNA from 250 to 400 bp, then pooled using the QIAgility liquid-handling robot. The pooled PCR products were cleaned up using the Mag-Bind RxnPure Plus (Omega Bio-tek) according to the manufacturer’s protocol. The cleaned pool was sequenced on the MiSeq (Illumina, Inc.) using the v2 2 × 250 base-pair kit (Illumina, Inc.). Positive/negative controls were included for DNA extraction and amplification.