Quick dna fecal soil microbe miniprep

Total DNA was isolated from fecal samples utilizing the Quick DNA Fecal/Soil Microbe Miniprep (Cat# D6010, ZYMO Research, Tustin, CA, USA) per the manufacturer’s instructions. Purified DNA was subjected to quantification and purity assessment via an Epoch microplate spectrophotometer (BioTek Instruments, Winooski, VT, USA).

Total microbial community DNA was extracted from all sample types and analyzed by using the Quick-DNA Fecal/Soil Microbe miniprep (Zymo Research Corp, Irvine, CA, USA) following manufacturer's recommendations. Pure DNA was quantified using a Qubit 2.0 fluorometer. DNA samples at a 5 ng/μl concentration were used to prepare libraries using the Illumina 16S-metagenomics library preparation protocol. The V3–V4 hyper variable region of bacterial 16S rRNA gene was amplified by using the primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GACTACHVGGGTATCTAATCC-3′) containing Illumina adaptors as illustrated in Klindworth et al. (19 (link)). The PCR amplicons were checked in agarose gels and purified using AMPure XP beads (Beckman Coulter Inc) as per manufacturer's recommendations. Purified amplicons were then indexed by using the Nextera UD index set (Illumina, San Diego, CA, USA). Libraries were quantified in triplicate using a Qubit 2.0 fluorometer with a dsDNA HS Assay kit (Invitrogen, Carlsbad, CA, USA), pooled at equal concentrations (4 nM) to generate equivalent number of raw reads, and diluted to a final concentration of 6 pM. Amplicon libraries were spiked with 5% PhiX control (Illumina, San Diego, CA, USA) according to manufacturer's recommendations. Samples were sequenced using a MiSeq Reagent kit v3 (600 cycle) on an Illumina MiSeq platform (Illumina, San Diego, CA, USA).