Lee et al. BMC Genomics 2011, 12:197 http://www.biomedcentral.com/1471-2164/12/197 Page 3 of 6 Figure 1 Nucleotide alignment of the Serva ILTV sequence and the concatenated ILTV reference sequence. Single vertical black lines indicate single nucleotide differences. Wider black boxes indicate larger regions of sequence differences, including areas where there are large gaps. Dashes indicate single base gaps in sequence alignments. The alignment was performed using Clustal W version 2.0. The locations and sizes of predicted ORFs and origins of DNA replication were annotated using the Geneious software package. The solid lines below the residue numbers indicate the locations of the internal and terminal repeat sequences. missing regions were identified in the published conca- tenated reference sequence. Missing regions of genomic sequence considerably hamper genomic assembly, so the Serva sequence assembled in this study represents a much improved reference sequence for future high throughput ILTV sequencing studies and comparative genomic analyses. Methods Virus strain This study utilised the chicken embryo origin live atte- nuated Serva vaccine strain of ILTV (Nobilis® ILT, Intervet), which has been recently introduced into Aus- tralia [16]. Virions were purified by Ficoll gradient cen- trifugation directly from commercial vaccine vials. Pelleted virions were washed and resuspended in TNE buffer (0.01 M Tris, 0.2 M NaCl, 1 mM EDTA, pH 7.4). High-throughput sequencing Following purification, total viral genomic DNA was extracted using the High Pure PCR Template Preparation Kit (Roche). Sequencing was performed using parallel, ligation-mediated sequencing technology (SOLiD™ 3 system, Applied Biosystems) following the manufacturer’s standard procedures. Briefly, 1 μg of ILTV DNA was sheared and P1 and P2 adaptors were ligated to the fragments. Ligated DNA fragments were size-selected to an average length of 170 bp and ampli- fied for 10 cycles. Approximately 10 pg of this ILTV fragment library/μl, as well as 50 pg of a similarly gen- erated library of an unrelated bacterial genome of around one megabase pairs/μl, were added to an emul- sion with 80 million beads. The libraries were sequenced in parallel using a flow cell divided into 8 segments. The resulting reads were unambiguously mapped to either the viral or the bacterial genome, allowing up to two mismatches for each read. The software package Velvet version 0.7.55 was used to perform de novo assembly of all reads [24] and the resulting contigs for the virus were identified bioinfor- matically and then aligned to the complete concate- nated ILTV genomic sequence, with the exception of