Shotgun-seq of Tursiops truncatus: adult serum sample
Animal fecal samples were weighed in 50 mL sterile conical tubes. Sterile phosphate-buffered saline (PBS) was added at 1 mL/g and vortexed for 5 minutes.  Sterile PBS was also used as a negative control.  The samples were centrifuged at 15,000 g for 10 minutes, and the supernatant was filtered through 0.45 m and 0.22 m filters.  Serum samples were filtered directly through 0.45 m and 0.22 m filters. In order to remove any free DNA, 1 mL aliquot of each sample was incubated with Ambion DNase I (RNase Free) (Thermo Fisher Scientific, USA) at 37oC for 1 hour in a water bath at a final concentration of 0.1 U (unit) per L. The DNase was deactivated by treating with 50 mM EDTA at 75oC in a heat block for 10 minutes. Viral nucleic acids were extracted using the Invitrogen PureLink Viral RNA/DNA Extraction kit according to manufacturers instructions (Thermo Fisher Scientific, USA). In this study, 200 L of pre-treated samples were extracted and eluted into 50 L sterile RNase-free water.  Nucleic acid samples were stored in -80C until further processing.  To generate sufficient material for sequencing, a random reverse transcription/amplification protocol was used to amplify both viral DNA and RNA. Briefly, 5 L of the nucleic acid samples were mixed with 1uL of primer-A (5-GTTTCCCAGTCACGATCNNNNNNNNN) (40M) (D. Wang et al., 2002; David Wang et al., 2003), 4 L nuclease-free water, and was heated to 65oC for 5 minutes and 22oC for 5 minutes. The 10 L reaction was then added to 4 L 5X First Strand Buffer [250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2 ], 0.4 L deoxynucleotide Solution Mix (dNTP) (10mM) (New England Biolabs, USA),  0.6 L nuclease-free water, 2 L DTT (100mM), 2uL SuperScript III Reverse Transcriptase (200U/uL) (Thermo Fisher Scientific, USA), 1uL RNaseOUT (40U/uL) (Thermo Fisher Scientific, USA) and was heated to 42oC for 60 minutes, 94oC for 2 minutes, and 10oC for 5 minutes.  The 20 L reaction was added to 2 L 5X Sequenase Reaction Buffer, 7.7 L nuclease-free water, 0.3 L Sequenase Version 2.2 DNA Polymerase (13 U/L) (Thermo Fisher Scientific, USA), and was heated to 10oC for 5 minutes, 37oC for 8 minutes with 1oC/s ramping, 94oC for 2 minutes, 10oC for 5 minutes (1.2L of 2.5 L Sequenase Version 2.2 DNA Polymerase (13 U/uL) with 7.5 L Sequenase Enzyme Dilution Buffer was added to each tube during this step), 37oC for 8 minutes with 1oC/s ramping, and 94oC for 8 minutes. 6 L of the cDNA template was added to 8 L MgCl2 , 10 L Buffer II (100mM Tris-HCl, pH 8.3, 500 mM KCl), 1 L dNTP (10mM), 1 L Primer-B (5-GTTTCCCAGTCACGATC) (100M) (David Wang et al., 2002; D. Wang et al., 2003), 1 L AmpliTaq Gold DNA Polymerase with Gold Buffer and MgCL2  (Thermo Fisher Scientific, USA), and 73 L nuclease-free water. The cDNA was amplified under the following conditions, 94oC for 15 minutes, 40 cycles of 94oC for 30 seconds, 40oC for 30 seconds, 50oC for 30 seconds, and 72oC for 1 minute.  The resulting PCR product was run on a 1% agarose gel and a smear between 500bp to 1kb was considered positive for sequencing.  Positive samples were purified using the Wizard SV Gel and PCR Clean-Up System according to manufacturer's instructions (Promega, Madison, WI, USA), where 100 L cDNA was purified and eluted into 50 L nuclease-free water.  Amplified samples were checked for DNA concentration using Qubit 4  dsDNA HS Assay kit according to manufacturers instructions (Thermo Fisher Scientific, USA), and 1 L samples were tested for concentration (ng/ L). 50 L of purified rPCR samples were barcoded using the Oxford Nanopore PCR Barcoding Kit (SQK-PBK004) Version: RPB_9059_v1_revN_14Aug2019 according to the manufacturers instructions (Oxford Nanopore, UK). Samples were sequenced on the MinION Mk1B with R9.4.1 flow cell using the MinKNOW software package v20.10.3 with Guppy basecaller v4.4.1 and MinKNOW v22.23.06 with Guppy basecaller v6.2.1.  The libraries were analyzed in the MinKNOW software under the following parameters: PCR Barcoding kit, Native Barcoding Expansion 1-12 (EXP-PBC001), fast basecalling, trim barcodes, mid-read barcode filtering and barcode trimming.