Truseq universal adapter

Individual samples were clustered in fifteen pools based on bird species, sample type (i.e., cloacal swab or sera), date and place of collection (Table 1), and the pools were processed as previously described^{49 (link)}. To remove naked DNA and RNA, 200 μl of the resuspended pellet from each pool were digested in a cocktail with 20U of Turbo DNase (Life Technologies, USA), 25U of benzonase (Sigma-Aldrich, USA), and 0.1 mg/ml of RNase A (Life Technologies, USA) at 37 °C for 2 hours in 20 μl of 10X DNase buffer (Life Technologies, USA). Subsequently, the viral genomes were extracted with a QIAamp viral RNA mini kit (Qiagen, Hilden, USA). cDNA was synthesized using Superscript II cDNA synthesis kit and random hexamers (Invitrogen, Carlsbad, USA) according to the manufacturer’s instructions. Then, cDNAs were prepared for high-throughput sequencing using TruSeq Universal adapters (Illumina, San Diego, USA) and standard multiplex adaptors. A paired-end, 150-base-read protocol in RAPID module was used for sequencing in an Illumina HiSeq 2500 instrument as recommended by the manufacturer. Sequencing was performed in the Life Sciences Core Facility (LaCTAD) at the State University of Campinas (UNICAMP), Brazil. Sequencing reads were de novo assembled using the MetaViC pipeline (available on https://github.com/sejmodha/MetaViC) as previously described^{50 (link)}.

The quantity of LR-PCR DNA templates (prior and during library preparation) was assessed by spectrophotometry (Nanodrop ND-800, Thermo Scientific, Waltham, MA, USA), fluorometry (Qubit, Invitrogen, Carlsbad, NM, USA) and quantitative PCR (PicoGreen dosage with Quant-iT™ PicoGreen® dsDNA Assay Kit, Invitrogen on ABI 7900HT, Applied Biosystems). After quality controls, six libraries (one per individual) were prepared using the TruSeq DNA Sample Prep Kits v2 and TruSeq Universal Adapters (Illumina, San Diego, CA, USA). A TruSeq Universal Adapter was used for each DNA library in order to separate reads from different individuals after DNA sequencing. Library sizes were checked on BioAnalyzer chips (Agilent Technologies, Santa Clara, CA, USA). Paired-end library sequencing was carried out on the Illumina MiSeq plateform (2 × 250 bp chemistry) at the GeT-PlaGe lab (GenoToul, Toulouse, France).