Kapa library preparation kit

Illumina library construction and sequencing were performed at the Genome Sequencing and Analysis Core Resource at Duke University. Indexed amplicon libraries were constructed using a KAPA Library Preparation Kit (KAPA Biosystems, USA). Libraries were pooled in equimolar concentrations and multiplex sequenced (150 bp paired-end) on a single flowcell using the Illumina MiSeq. To improve the data quality of low-diversity samples (i.e., single-fragment PCR products such as ours), the run included a 30–50% PhiX DNA spike-in control. Raw reads are available by sample in the NCBI Sequence Read Archive (SRA; accessions SRX375380-375387).

PCR products were purified using QIAquick PCR purification kit (QIAGEN). Ion Torrent deep sequencing libraries were constructed from bisulfite-converted DNA using the KAPA Library Preparation Kit (Kapa Biosystems), quantified using the QIAxcel Advanced System (QIAGEN), and templated using the Ion PGM Template OT2 200 kit (Thermo Fisher). The libraries were sequenced using an Ion PGM Sequencing HiQ Kit with Ion 316 v2 Chips on the Ion Torrent PGM (Thermo Fisher), which generated non-directional ~200 nt length reads, 1500–7500 reads per library in the fastq format. Reads were aligned to human or mouse mtDNA reference sequences (hg38 and mm10, respectively) using the Bismark aligner [25 (link)] with bowtie2 option. Bisulfite conversion efficiency was evaluated based on cytosine conversion rate (C versus T) at non-CpG sites determined after visualization of the aligned reads (bam format) using the Integrative Genomics Viewer (IGV) [18 (link)].

The isolates were recovered in Madin-Darby canine kidney cells (American Type Culture Collection, Manassas, VA, USA) by using standard cell culture techniques, and cell-released virus was pelleted from the infected cell medium at a late stage of infection by ultracentrifugation at 145,500 g for 3 h. The pellet was resuspended in 200 μl 10 mM Tris-HCl (pH 8) and centrifuged at 900 g for 3 min to pellet cellular debris. DNA was extracted from the supernatant by using a DNeasy blood and tissue kit (Qiagen, Crawley, UK), and quantified by using a Qubit 2.0 fluorometer (Life Technologies Co., Carlsbad, CA, USA). Aliquots of DNA from CHV/0194, CHV/V777 and CHV/V1154 (0.565 μg, 0.605 μg and 0.336 μg, respectively) were sheared acoustically to an average size of 460 nucleotides (nt) in a volume of 50 μl by using a Covaris S220 sonicator (Covaris Inc., Woburn, MA, USA). Fragment size was measured by using an Agilent 2200 Tapestation (Agilent, Santa Clara, CA, USA). A KAPA library preparation kit (KAPA Biosystems, Wilmington, MA, USA) was used to prepare the sheared DNA fragments for Illumina sequencing, as described previously [31 (link)].

Genomic DNA for long- and short-read sequencing was extracted by using a DNeasy PowerSoil kit (Qiagen, Hilden, Germany). Short-read sequencing was conducted on an Illumina HiSeq 3000 sequencer using the KAPA library preparation kit (Kapa Biosystems, Woburn, MA) or on an Illumina MiSeq sequencer using the KAPA HyperPlus Library Preparation kit (Kapa Biosystems). Long-read sequencing was conducted on a Nanopore GridION sequencer (Oxford Nanopore Technologies, Oxford, UK) using sn SQK-LSK109 1D ligation sequencing kit and sn EXP-NBD103 native barcoding kit. The reads were assembled and polished using Unicycler (37 (link)). In cases where the complete plasmid sequences could not be constructed, sequences were assembled with CANU (version 1.8) (38 (link)) or flye (39 (link)) and improved using Pilon (40 (link)) or Racon (41 (link)). The PlasmidFinder (42 (link)) and ResFinder (43 (link)) databases were used to identify antimicrobial resistance genes and plasmid replicon types, respectively. A detailed analysis of the insertion sequence was performed using ISfinder (44 (link)). The sequences were annotated with RASTtk (45 (link)), and the genomic structures were compared with EasyFig (46 (link)). Plasmids similar to those found in this study were identified using BLAST.

Library preparation of 42 specimens (including all cell culture isolates) with sufficient HAdV DNA load (a ratio of HAdV/human genomic DNA >1,000) was performed using Nextera XT DNA library preparation kit (Illumina, San Diego, CA, USA) with 1 ng of DNA as input following manufacturers’ guideline. Purified DNA of the other nine specimens (6C1, 19C2, 20C1, 23C2, 24C2, 35C2, 47C2, 48C2 and 50C1) was sheared by sonication, and sequencing libraries were prepared directly (KAPA library preparation kit, KAPA Biosystems, Wilmington, MA, USA) as previously described^{49 (link)}, with a few modifications. DNA fragments were end-repaired, A-tailed and ligated to NEBnext Illumina adapter (New England Biolabs, Ipswich, MA, USA). After PCR pre-amplification (6–14 cycles) with adapter specific primers, up to 750 ng of DNA was target enriched for HAdV fragments with RNA baits. These 120-mer RNA baits were designed to span the length of the positive strand of 24 Genbank HAdV-C1, -C2 and -C5 reference genomes (SureSelectXT kit, Agilent Technologies, CA, USA).