Research output: Contribution to Journal/Magazine › Journal article › peer-review
Research output: Contribution to Journal/Magazine › Journal article › peer-review
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TY - JOUR
T1 - Sequences of complete human cytomegalovirus genomes from infected cell cultures and clinical specimens
AU - Cunningham, Charles
AU - Gatherer, Derek
AU - Hilfrich, Birgitta
AU - Baluchova, Katarina
AU - Dargan, Derrick J.
AU - Thomson, Marian
AU - Griffiths, Paul D.
AU - Wilkinson, Gavin W. G.
AU - Schulz, Thomas F.
AU - Davison, Andrew J.
PY - 2010/3/1
Y1 - 2010/3/1
N2 - We have assessed two approaches to sequencing complete human cytomegalovirus (HCMV) genomes (236 kbp) in DNA extracted from infected cell cultures (strains 3157, HAN13, HAN20 and HAN38) or clinical specimens (strains JP and 3301). The first approach involved amplifying genomes from the DNA samples as overlapping PCR products, sequencing these by the Sanger method, acquiring reads from a capillary instrument and assembling these using the Staden programs. The second approach involved generating sequence data from the DNA samples by using an Illumina Genome Analyzer (IGA), processing the filtered reads by reference-independent (de novo) assembly, utilizing the resulting sequence to direct reference-dependent assembly of the same data and finishing by limited PCR sequencing. Both approaches were successful. In particular, the investigation demonstrated the utility of IGA data for efficiently sequencing genomes from clinical samples containing as little as 3 % HCMV DNA. Analysis of the genome sequences obtained showed that each of the strains grown in cell culture was a mutant. Certain of the mutations were shared among strains from independent clinical sources, thus suggesting that they may have arisen in a common ancestor during natural infection. Moreover, one of the strains (JP) sequenced directly from a clinical specimen was mutated in two genes, one of which encodes a proposed immune-evasion function, viral interleukin-10. These observations imply that HCMV mutants exist in human infections.
AB - We have assessed two approaches to sequencing complete human cytomegalovirus (HCMV) genomes (236 kbp) in DNA extracted from infected cell cultures (strains 3157, HAN13, HAN20 and HAN38) or clinical specimens (strains JP and 3301). The first approach involved amplifying genomes from the DNA samples as overlapping PCR products, sequencing these by the Sanger method, acquiring reads from a capillary instrument and assembling these using the Staden programs. The second approach involved generating sequence data from the DNA samples by using an Illumina Genome Analyzer (IGA), processing the filtered reads by reference-independent (de novo) assembly, utilizing the resulting sequence to direct reference-dependent assembly of the same data and finishing by limited PCR sequencing. Both approaches were successful. In particular, the investigation demonstrated the utility of IGA data for efficiently sequencing genomes from clinical samples containing as little as 3 % HCMV DNA. Analysis of the genome sequences obtained showed that each of the strains grown in cell culture was a mutant. Certain of the mutations were shared among strains from independent clinical sources, thus suggesting that they may have arisen in a common ancestor during natural infection. Moreover, one of the strains (JP) sequenced directly from a clinical specimen was mutated in two genes, one of which encodes a proposed immune-evasion function, viral interleukin-10. These observations imply that HCMV mutants exist in human infections.
UR - http://www.scopus.com/inward/record.url?scp=76849105203&partnerID=8YFLogxK
U2 - 10.1099/vir.0.015891-0
DO - 10.1099/vir.0.015891-0
M3 - Journal article
AN - SCOPUS:76849105203
VL - 91
SP - 605
EP - 615
JO - Journal of General Virology
JF - Journal of General Virology
SN - 0022-1317
IS - 3
ER -