Viral metagenome in chicken cloaca swabs:ch01
In Nov. 2017, 40 cloaca swabs samples were collected form 40 apparently healthy chickens in two commercial chicken farm in Anhui province in China. These samples were then subjected to viral nucleic acid detection using viral metagenomic method. All fecal samples were collected by disposable materials and shipped on dry-ice. The swabs were dipped in Dulbecco phosphate buffered saline (DPBs) with the volume of 0.5 ml and vortexed vigorously for 10 minutes and then were centrifuged at 15,000  g for 10 minutes. 100 L of each supernatant was then collected and pooled into a single sample pool. The pooled supernatant was filtered through a 0.45-mm filter (Millipore) to remove eukaryotic and prokaryotic particles, then subjected to a mixture of nuclease enzymes to reduce the concentration of non-viral encapsidated nucleic acids. The total nucleic acid was extracted using QIAamp MinElute Virus Spin Kit (Qiagen) according to manufacturer's instructions. Reverse transcription reaction using reverse transcriptase (Super-Script IV, Invitrogen) was then performed based on the enriched viral nucleic acid preparation and 100 pmol of random hexamerprimer, followed by a round of DNA synthesis using Klenow fragment polymerase (New England BioLabs). Library construction was then constructed using Nextera XT DNA Sample Preparation Kit (Illumina) and sequenced on the Illumina MiSeq platform with 250 bases paired ends with dual barcoding.