The current methodology comprises two steps: (1) purification of the microbial cells from fecal impurities and (2) lysis of microbial cells to obtain metagenomic DNA with high molecular weight.
At the first step, fresh feces (100 mg) were weighed into a sterile microcentrifuge tube for isolation of purified microbial cells. Microbial cells were sequentially washed with normal saline solution (0.9% NaCl solution) and phosphate-buffered saline (PBS; pH 7.4). The washing steps were optimized for the recovery of a purified bacterial pellet to obtain quality fecal metagenomic DNA for the downstream studies. In 5 sets of replicates, 100 mg of feces were resuspended in 1 ml of normal saline solution by vortexing for 30 s and then centrifuged at ambient room temperature (RT) for 2 min with different speed of 1000 rpm (72 × g), 2000 rpm (287 × g), 3000 rpm (645 × g), 4000 rpm (1147 × g), and 5000 rpm (1792 × g), respectively. The resulting supernatants were subjected to microscopic examination for the presence of fibers and insoluble impurities. Recovered supernatant was centrifuged again at 10,000 rpm (7168 × g) for 1 min at ambient room temperature to collect microbial pellet for downstream processing. Microbial pellet from all replicates was subsequently washed with 1 ml of PBS (pH 7.4) for centrifugation with different speeds as described above. The resulting supernatants were subjected to centrifugation again at 10,000 rpm (7168 × g) for 1 min to recover microbial pellet, which was used for metagenomic DNA isolation at the next step.
At the second step, the purified microbial pellet was resuspended in 500 μl of lysis buffer containing 1% (w/v) cetyl trimethyl ammonium bromide (CTAB), 100 mM of ethylenediaminetetraacetic acid (EDTA), 1.5 M of NaCl, 100 mM of Na3PO4, and 100 mM of Tris–HCl (pH 8.0). After adding 2 μl of proteinase K (20 mg/ml), the mixture was incubated for 10 min at 37 °C with gentle shaking at 100 rpm in orbital shaker incubator. Afterward, sodium dodecyl sulfate (SDS) was added with a final concentration of 1% and the incubation continued for another 20 min at 65 °C with intermittent shaking. The lysate was centrifuged at 13,000 rpm (12,114 × g) for 5 min at ambient room temperature. The resulting supernatant was collected and mixed with an equal volume of saturated phenol:chloroform:isoamyl alcohol (25:24:1), which is then subjected to centrifugation at 10,000 rpm (7168 × g) for 5 min at RT. The aqueous phase was collected and metagenomic DNA was precipitated with 0.6 volume of isopropanol and pelleted by centrifugation at 13,000 rpm (12,114 × g) for 5 min. After washing twice with 70% ethanol, the resulting DNA was dried and finally dissolved into a 50 μl of 1 × Tris–EDTA buffer (pH 8.0).
The qualitative and quantitative analysis of the metagenomic DNA was performed by agarose gel electrophoresis, restriction endonuclease digestion (Sau3A1), NanoQuant (Tecan Group, Mannedorf, Switzerland) estimation, and Qubit® dsDNA HS Assay Kit (Life Technologies, Carlsbad, CA). Metagenomic DNA recovered from all replicates was compared for qualitative and quantitative parameters to obtain optimized condition for metagenomic DNA isolation from human feces. The optimized method is outlined inFigure 1 and was then used to isolate the metagenomic DNA from 10 one-month-old frozen feces stored at −86 °C and 50 random fecal samples (including both fresh and frozen samples) to evaluate its robustness.
At the first step, fresh feces (100 mg) were weighed into a sterile microcentrifuge tube for isolation of purified microbial cells. Microbial cells were sequentially washed with normal saline solution (0.9% NaCl solution) and phosphate-buffered saline (PBS; pH 7.4). The washing steps were optimized for the recovery of a purified bacterial pellet to obtain quality fecal metagenomic DNA for the downstream studies. In 5 sets of replicates, 100 mg of feces were resuspended in 1 ml of normal saline solution by vortexing for 30 s and then centrifuged at ambient room temperature (RT) for 2 min with different speed of 1000 rpm (72 × g), 2000 rpm (287 × g), 3000 rpm (645 × g), 4000 rpm (1147 × g), and 5000 rpm (1792 × g), respectively. The resulting supernatants were subjected to microscopic examination for the presence of fibers and insoluble impurities. Recovered supernatant was centrifuged again at 10,000 rpm (7168 × g) for 1 min at ambient room temperature to collect microbial pellet for downstream processing. Microbial pellet from all replicates was subsequently washed with 1 ml of PBS (pH 7.4) for centrifugation with different speeds as described above. The resulting supernatants were subjected to centrifugation again at 10,000 rpm (7168 × g) for 1 min to recover microbial pellet, which was used for metagenomic DNA isolation at the next step.
At the second step, the purified microbial pellet was resuspended in 500 μl of lysis buffer containing 1% (w/v) cetyl trimethyl ammonium bromide (CTAB), 100 mM of ethylenediaminetetraacetic acid (EDTA), 1.5 M of NaCl, 100 mM of Na3PO4, and 100 mM of Tris–HCl (pH 8.0). After adding 2 μl of proteinase K (20 mg/ml), the mixture was incubated for 10 min at 37 °C with gentle shaking at 100 rpm in orbital shaker incubator. Afterward, sodium dodecyl sulfate (SDS) was added with a final concentration of 1% and the incubation continued for another 20 min at 65 °C with intermittent shaking. The lysate was centrifuged at 13,000 rpm (12,114 × g) for 5 min at ambient room temperature. The resulting supernatant was collected and mixed with an equal volume of saturated phenol:chloroform:isoamyl alcohol (25:24:1), which is then subjected to centrifugation at 10,000 rpm (7168 × g) for 5 min at RT. The aqueous phase was collected and metagenomic DNA was precipitated with 0.6 volume of isopropanol and pelleted by centrifugation at 13,000 rpm (12,114 × g) for 5 min. After washing twice with 70% ethanol, the resulting DNA was dried and finally dissolved into a 50 μl of 1 × Tris–EDTA buffer (pH 8.0).
The qualitative and quantitative analysis of the metagenomic DNA was performed by agarose gel electrophoresis, restriction endonuclease digestion (Sau3A1), NanoQuant (Tecan Group, Mannedorf, Switzerland) estimation, and Qubit® dsDNA HS Assay Kit (Life Technologies, Carlsbad, CA). Metagenomic DNA recovered from all replicates was compared for qualitative and quantitative parameters to obtain optimized condition for metagenomic DNA isolation from human feces. The optimized method is outlined in
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