Lee et al. BMC Genomics 2011, 12:197 http://www.biomedcentral.com/1471-2164/12/197 fold. This depth of coverage exceeds the level required for precision whole-genome sequencing and demon- strates the suitability of the massively parallel, ligation- mediated sequencing method for herpesvirus genome sequencing [14]. Although the depth of coverage was sufficient to generate long contigs able to cover the whole genome, a total of 185 gaps or regions of ambigu- ous sequence were detected after de novo assembly. Most gaps were less than 100 bp in size. While sonica- tion theoretically produces random fragment libraries, some sequences, such as A-T rich regions, have been found to be more susceptible to breakage [15]. Such weak regions may have been cleaved more frequently during our fragment library preparation, resulting in an absence of high-throughput sequencing data across these regions. Overview of the Serva ILTV genome The general features of the Serva ILTV genome sequence were examined and were consistent with those described in previous genome studies of ILTV [3-12]. The genome size of the Serva ILTV strain was 152,630 bp, with a G + C content of 48%. The unique long and unique short sequences were 113,930 bp and 13,094 bp in length, respectively, with the unique short sequence flanked by internal and terminal repeat sequences (each 12,803 bp in length). A total of 80 predicted open read- ing frames (ORFs) were identified. Sixty-five ORFs were located within the long unique region, nine within the unique short region, and three pairs of ORFs within the repeat regions of the genome. An annotated genome map of the Serva ILTV strain is shown in Figure 1. Comparison of the Serva ILTV genome with the concatenated ILTV reference sequence Comparative analyses showed that the Serva strain had the same gene arrangement as that of the concatenated ILTV genomic reference sequence (Figure 1). In com- plete genomic alignment analysis, the Serva strain had 96.5% DNA sequence identity with the concatenated sequence. Differences between the two sequences were mostly located within the left terminal region of the genome (extending over 18,169 bp), the middle of the unique long region of the genome (extending over 31,332 bp) and within the repeat regions (extending over 8,364 bp) (Figure 1). In the concatenated reference sequence, all these regions were obtained from SA-2 ILTV, a commercial vaccine strain of ILTV produced from an Australian field isolate [13]. Australian strains of ILTV may contain different genetic features com- pared with other ILTV strains, due to their evolution in a geographically isolated environment [16]. Excluding SA-2 sequence regions, DNA sequence identity between the Serva strain sequenced in this study and the Page 2 of 6 concatenated sequence was 99.9%, containing 41 single- nucleotide polymorphisms and 13 nucleotide insertions or deletions. This high level of identity is consistent with the stable genome and low mutation rates observed in herpesviruses [17,18]. All nucleotide differences between the concatenated reference and the Serva strain of ILTV are listed in Additional File 1. Four large regions of sequence were absent in the con- catenated ILTV sequence compared with the Serva strain. These included 528 bp and 594 bp of sequence missing in the UL29 and UL36 genes, respectively. Addi- tionally, in each of the repeat regions the concatenated sequence lacked 1,563 bp of sequence compared to the Serva strain. To verify these findings, PCR was used to amplify the relevant regions of the UL29 and UL36 genes, and the repeat region of the Serva genome. The PCR products were sequenced using Sanger sequencing. The results obtained from Sanger sequencing were iden- tical to those obtained by the high-throughput sequen- cing. The predicted translation products of the UL29 and UL36 genes of the Serva strain more closely resembled, and had more conserved domains when compared with, the UL29 and UL36 homologues from other alphaherpes- viruses than the predicted products derived from the concatenated genome sequence (Figure 2). These results support the conclusion that the newly determined addi- tional sequences are genomic sequence of ILTV, not sequencing errors. Within the left terminal region of the genome, the Serva sequence had a 189 bp deletion com- pared to the concatenated sequence (Figure 1). There were differences in size of more than 50 amino acids between the predicted translation products of the UL54, UL29, UL30, UL36, UL37 and UL38 genes in the concatenated reference sequence and the Serva sequence. Smaller differences in the predicted sizes of other gene products were also identified (Table 1). All amino acid differences between predicted translation products in the concatenated reference sequence and the Serva sequence are listed in Additional File 2. Given the conserved nature of the ILTV genome, future studies examining the genomic variation between different strains of ILTV may represent a strategic approach to examining the molecular pathogenesis of this virus. In particular it would be useful to compare sequence differences between virulent and attenuated strains of ILTV, especially within genes already known to be associated with ILTV virulence including gC, UL0, gG, gJ and TK genes [19-23]. Conclusions This is the first complete genomic sequence of an indi- vidual ILTV strain. A number of differences between this strain and the concatenated reference ILTV sequence were identified. Significantly, four large