v3-v4 region of 16S rRNA gene |
Total DNA of rumen fluid and the feces was extracted from individual samples using a stool DNA extraction kitTM (Qiagen, Hilden, Germany). The 16S universal primers 338 F (5?- ACTCCTACGGGAGGCAGCAG-3?) and 806 R (5?-GGACTACHVGGGTWTCTAAT-3?) were used to amplify 16S rRNA genes (regions V3-V4). Following ampli?cation, PCR products were analyzed on a 2% agarose gel to con?rm correct product size, and were purified with AxyPrep DNA Purification kit (Axygen Biosciences, Union City, USA). The purified PCR products were quantitatively determined using QuantiFluor-ST Fluoremeter (Promega, Wisconsin, USA) and PicoGreen dsDNA Quantitation Reagent (Invitrogen, Carlsbad, USA), and then were pooled in equimolar and paired-end sequenced (2×300) on an Illumina MiSeq platform (San Diego, CA, United States) according to the standard protocols. |
WGS |
METAGENOMIC |
PCR |
PAIRED
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