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Total genomic DNA from feed, milk and colonic contents samples (SPF and GF piglets) were extracted using a QIAGEN kit, and quantified using a Qubit 2.0 (Invitrogen, USA). The V3-V4 hypervariable regions of the 16S rDNA gene were amplified by universal primers with barcode. PCR products was purified with GeneJET Gel Extraction Kit (Thermo Scientific). Sequencing libraries were generated using TruSeq DNA PCR-Free Library Preparation Kit (Illumina, USA) following manufacturer’s recommendations, and were sequenced using the 250 bp paired-end strategy on an Illumina NovaSeq platform. |
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size fractionation |
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