Metagenomic analysis of Sichuan takin fecal sample viromes
The viral transport medium containing the 71 fecal samples were pooled, 200  l (about 200 mg of feces per 2 ml viral transport medium) each, and centrifuged at 10000  g for 10 min. The supernatant was then filtered through a 0.45- m filter to remove eukaryotic and bacterial cell-sized particles. The filtrate was treated with a cocktail of DNase enzymes, including 14 U of turbo DNase, 20 U of benzonase and 20 U of RNase One at 37 C for 60 min in 1  DNase buffer to digest unprotected nucleic acids. Total RNA from the sample was extracted using the QIAamp Viral RNA Mini Kit. Reverse transcription was performed using SuperScript III reverse transcriptase and a random primer containing a 20-base arbitrary sequence at the 5  end followed by a randomized octamer (8N) at the 3  end. A single round priming and extension was performed using Klenow fragment polymerase. PCR amplification with primer consisting of 20-base arbitrary sequence of the random primer was performed in 20 cycles of 94 C for 15 s, 60 C for 30 s and 68 C for 1 min and a final extension at 68 C for 7 min in an automated thermal cycler Standard precautions were taken to avoid PCR contamination and no amplified PCR product was observed in negative control. The PCR product was purified using the MinElute PCR Purification Kit following the manufacturer s protocol with slight modification. The purified DNA was eluted in 15  l of EB buffer and used as the template for library construction. The metagenomic library was prepared using Nextera XT DNA Sample Preparation Kit and Nextera XT Index kit according to the manufacturer s protocol. Briefly, 1 ng of input DNA was tagmented by the Nextera XT transposome at 55 C for 5 min. This transposome simultaneously fragmented the input DNA and added adaptor sequence to the ends, allowing amplification by PCR in subsequent steps. The sequencing library with tagmented DNA was then amplified in 12 cycles of 95 C for 10 s, 55 C for 30 s and 72 C for 30 s, and a final extension at 72 C for 5 min in an automated thermal cycler. Amplified DNA library was purified using 1.8   AMPure XP beads to remove very short library fragments from the population. Purified sequencing library was quantified using KAPA library quantification kit and subsequently sequenced on Illumina HiSeq 2500 with 151 bp paired-end reads (Rapid Run Mode). Image analysis and base calling were performed with SCS2.8/RTA1.8. FASTQ file generation and the removal of failed reads were performed using CASAVA ver.1.8.2.