Development and Application of a Fast Method to Acquire the Accurate Whole-Genome Sequences of Human Adenoviruses

Abstract

The whole-genome sequencing (WGS) of human adenoviruses (HAdVs) plays an important role in identifying, typing, and mutation analysis of HAdVs. Nowadays, three generations of sequencing have been developed. The accuracy of first-generation sequencing is up to 99.99%, whereas this technology relies on PCR and is time consuming; the next-generation sequencing (NGS) is expensive and not cost effective for determining a few special samples; and the third-generation sequencing technology has a higher error rate. In this study, first, we developed an efficient HAdV genomic DNA extraction method. Using the complete genomic DNA instead of the PCR amplicons as the direct sequencing template and a set of walking primers, we developed the HAdV WGS method based on first-generation sequencing. The HAdV whole genomes were effectively sequenced by a set of one-way sequencing primers designed, which reduced the sequencing time and cost. More importantly, high sequence accuracy is guaranteed. Four HAdV strains (GZ01, GZ02, HK35, and HK91) were isolated from children with acute respiratory diseases (ARDs), and the complete genomes were sequenced using this method. The accurate sequences of the whole inverted terminal repeats (ITRs) at both ends of the HAdV genomes were also acquired. The genome sequence of human adenovirus type 14 (HAdV-B14) strain GZ01 acquired by this method is identical to the sequence released in GenBank, which indicates that this novel sequencing method has high accuracy. The comparative genomic analysis identified that strain GZ02 isolated in September 2010 had the identical genomic sequence with the HAdV-B14 strain GZ01 (October 2010). Therefore, strain GZ02 is the first HAdV-B14 isolate emergent in China (September 2010; GenBank acc no. MW692349). The WGS of HAdV-C2 strain HK91 and HAdV-E4 strain HK35 isolated from children with acute respiratory disease in Hong Kong were also determined by this sequencing method. In conclusion, this WGS method is fast, accurate, and universal for common human adenovirus species B, C, and E. The sequencing strategy may also be applied to the WGS of the other DNA viruses.

Keywords: Sanger sequencing; genomic DNA; human adenovirus; human adenovirus type 14; human adenovirus type 2; human adenovirus type 4; inverted terminal repeats (ITRs); whole-genome sequencing.

FIGURE 1

Human adenovirus (HAdV) genome sequencing strategy. The genomic DNA extracted and purified is sent to a sequencing company along with all the walking primers. Red arrows indicate additional sequencing cases that may occur.

FIGURE 2

Agarose gel electrophoresis of the genomic DNA of four adenovirus isolates (HK91, HK35, GZ01, and GZ02) and restriction enzyme analysis of the genomes (A) in silico and (B) wet bench. MW, λ DNA/HindIII marker; lane 1, HK91 genome; 2, HK35 genome; 3, GZ01 genome; 4, GZ02 genome; 5–8, four genomes + EcoRI digestion. The predicted molecular weights of digested fragments of HK91 are 21,360, 4,294, 3,673, 2,670, 2,218, and 1,739 bp; of HK35 are 25,289, 6,404, and 4,273 bp; and of GZ01 and GZ02 are 21,917, 7,032, and 5,815 bp.

FIGURE 3

Inverted terminal repeat (ITR) alignment between reference strains and clinical isolates (A) HK91, (B) GZ01 and GZ02, and (C) HK35. HAdV-C2 reference strain: A8649 (JX173077); HAdV-B14 reference strain, deWit (AY803294); HAdV-E4 reference strain, ZG (KX384951). Different colors represent different base types.

FIGURE 4

Transcriptional maps and genome organizations of HAdV-B14 isolates (A) GZ01/GZ02, (B) HAdV-C2 isolate HK91, and (C) HAdV-E4 isolate HK35. The genome is indicated by two black horizontal lines marked at 10,000-bp intervals. The gray arrows indicate the early, intermediate, and late transcription units; the magenta arrows indicate coding regions. Arrows reflect the direction of the coding transcripts.

FIGURE 5

Phylogenetic analysis of the (A) whole genomes, (B) fiber, (C) hexon, and (D) penton base genes. Nucleotide sequences are from prototype strains in different types available from GenBank except for the four isolates (★). Taxon names include GenBank accession number, isolation country, strain name, and year of isolation. Phylogenetic trees were generated using the neighbor-joining method with 1,000 replicates and constructed by the MEGA X software. In these analyses, default parameters were applied, with a maximum-composite-likelihood model. Bootstrap numbers shown at the nodes indicate the percentages of 1,000 replications producing the clade, with values above 80 considered robust. The scale bar is in units of nucleotide substitutions per site.