Mo bio power fecal dna isolation kit

DNA was extracted using the MoBio PowerFecal DNA isolation kit (Mobio Laboratories, Carlsbad, CA, USA) according to the manufacturer’s protocol. The DNA extracts was used for shotgun sequencing using the Illumina Novaseq 6000. The raw data using Readfq V8 (https://github.com/cjfields/readfq) was preprocessed to acquire clean data for subsequent analyses. Sequencing data were deposited in the Genome Sequence Archive repository under BioProject number CRA005191.

A frozen aliquot (500 mg) of each fecal sample was processed, and bacterial DNA was extracted using the MO BIO PowerFecal™ DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, USA) by following the manufacturer's protocol. The DNA concentration (ranging from 15.2 to 75.4 ng/μl) of all samples was measured by NanoDrop (Thermo Scientific) and its quality was estimated on agarose gel electrophoresis. Only samples that meet the following criteria were used for library preparation: (1) DNA concentration is >15 ng/μl; (2) the total quantity of DNA is >6 μg; (3) DNA band that was visualized on agarose gel electrophoresis must be clear and of good quality. Finally, 1 μg DNA of each sample was pooled to yield an equimolar concentration to construct the DNA libraries (DNA was sheared to 350 bp) using the Illumina DNA Sample Preparation kit according to the manufacturer's instructions. Amplified libraries were sequenced on Illumina HiSeq 2500 instrument using paired-end 2 × 250 bp chemistry which was performed by Novogene (Beijing, China).
The metagenome dataset used in this study was deposited into the National Centre for Biotechnology Information's Sequence Read Archive (SRA; http://www.ncbi.nlm.nih.gov/sra) under accession bioproject number: PRJNA407583 (SRA number: SAMN07660490 - SAMN07660503).

Following the method of Jin et al. [22 (link)], 50 g of each fresh feces were prepared for DNA extraction and amplicon sequencing. In brief, the total microbial genomic DNA was extracted by means of the MoBio Power Fecal DNA Isolation Kit (MoBio Laboratories, Inc., Carlsbad, CA, USA) according to the manufacturer’s instructions. Successful DNA isolation was confirmed by agar gel electrophoresis. Bacterial amplicon libraries were prepared by amplifying the V4 region of 16S rRNA. Sequencing was performed using a 250-bp paired-end sequencing protocol on the Illumina MiSeq 2500 platform (Illumina, San Diego, CA, USA). The extraction kits and reagents in this study were used as negative (blank) controls, and no contaminant sequences were detected.

Bacterial genomic DNA was extracted using the Mo Bio Power Fecal DNA Isolation kit (Mo Bio Laboratories) according to the manufacturer’s instructions for high yields of DNA. To increase the DNA yields, the following modifications were used. An additional bead beater step using the Faster Prep FP120 (Thermo) at 6 m/s for 1 min was used instead of vortex agitation. Incubation with buffers C2 and C3 was increased to 10 min at 4 °C. Starting nucleic acid concentrations were determined by a Qubit Fluromoter (Life Technologies). A multiplexed amplicon library covering the 16S rDNA gene V4 region was prepared using the protocol of [53 (link)] with dual-index barcodes. The aggregated library pool was size selected from 300–500 bp on a pippin prep 1.5 % agarose cassette (Sage Sciences) according to the manufacturer’s instructions. The concentration of the pool was measured by qPCR (Kapa Biosystems) and loaded onto the MiSeq Illumina instrument (300 bp kit) at 6–9 pM with 40 % phiX spike-in to compensate for low base diversity according to Illumina’s standard loading protocol. Sequences were deposited in the NIH Sequence Read Archive (SRA accession SRP065075).

Total DNA was extracted by bead beating from approximately 50 mg of each fecal sample using the MOBIO PowerFecal DNA Isolation Kit (MOBIO Laboratories, Carlsbad, CA, USA) according to the manufacturer's protocol. DNA isolated from fecal samples was quantified using a NanoDrop (ThermoScienctific), and the V5–V6 regions of the 16S rRNA gene were PCR amplified using Accura High Fidelity Polymerase, with the addition of barcodes for multiplexing. The forward and reverse primers were the V5F and V6R sets [68 (link)], chosen in part to allow dual coverage of the entire region. The barcoded amplicons were pooled and Illumina adapters were ligated to the reads. A single lane on an Illumina MiSeq instrument was used (250 cycles, 300 bp, paired-end) to generate 16S rRNA gene sequences yielding 175,784 Pass Filter (PF) reads per fecal sample (SD = 72,822) and ~12.65 million total PF reads (4.9Gb of data). Raw sequencing data (fastq files) are available through MG-RAST [Project ID: mgp15238].