Next-generation sequencing for cytomegalovirus antiviral resistance genotyping in a clinical virology laboratory. Chorlton, S. D., Ritchie, G., Lawson, T., McLachlan, E., Romney, M. G., Matic, N., & Lowe, C. F. Antiviral Research, 192:105123, August, 2021. ![Next-generation sequencing for cytomegalovirus antiviral resistance genotyping in a clinical virology laboratory [pdf]](https://bibbase.org/img/filetypes/pdf.svg "pdf")
Paper doi abstract bibtex Introduction The identification of CMV antiviral drug resistance (AVDR) is a critical diagnostic test for immunocompromised patients with CMV infection and a failure of virologic response on optimal antiviral treatment. We developed a next-generation sequencing (NGS) assay for CMV AVDR testing and compared the AVDR mutations identified by NGS to Sanger sequencing. Methods Retrospective review of CMV AVDR testing requests for UL97 and UL54 at our laboratory from 2014 to 2019 was conducted. NGS was performed on the MinION and compared to Sanger sequencing performed at the national reference laboratory. Analysis of the sequences was completed with a novel cloud bioinformatics platform (BugSeq). Results Twenty patient samples previously characterized were included for study on the MinION. NGS captured all of the CMV AVDR mutations identified by Sanger, and identified additional mutations in UL97 and/or UL54 in 8/13 (62%) of the samples. An analysis of the depth of coverage at which we no longer detected minority single nucleotide variants (SNVs) detected in the original data was conducted, estimating a recall of 95% at 1800 fold coverage. Conclusion NGS utilizing MinION technology for the detection of CMV AVDR mutations identified additional minority variants in UL97 and UL54 as compared with Sanger sequencing. Through the application of a bioinformatics pipeline available online, our NGS process eliminates barriers associated with the use of the MinION and NGS in clinical laboratories.
@article{chorlton_next-generation_2021,
title = {Next-generation sequencing for cytomegalovirus antiviral resistance genotyping in a clinical virology laboratory},
volume = {192},
issn = {0166-3542},
url = {https://samchorlton.com/papers/NGS_CMV_AVDR.pdf},
doi = {10.1016/j.antiviral.2021.105123},
abstract = {Introduction
The identification of CMV antiviral drug resistance (AVDR) is a critical diagnostic test for immunocompromised patients with CMV infection and a failure of virologic response on optimal antiviral treatment. We developed a next-generation sequencing (NGS) assay for CMV AVDR testing and compared the AVDR mutations identified by NGS to Sanger sequencing.
Methods
Retrospective review of CMV AVDR testing requests for UL97 and UL54 at our laboratory from 2014 to 2019 was conducted. NGS was performed on the MinION and compared to Sanger sequencing performed at the national reference laboratory. Analysis of the sequences was completed with a novel cloud bioinformatics platform (BugSeq).
Results
Twenty patient samples previously characterized were included for study on the MinION. NGS captured all of the CMV AVDR mutations identified by Sanger, and identified additional mutations in UL97 and/or UL54 in 8/13 (62\%) of the samples. An analysis of the depth of coverage at which we no longer detected minority single nucleotide variants (SNVs) detected in the original data was conducted, estimating a recall of 95\% at 1800 fold coverage.
Conclusion
NGS utilizing MinION technology for the detection of CMV AVDR mutations identified additional minority variants in UL97 and UL54 as compared with Sanger sequencing. Through the application of a bioinformatics pipeline available online, our NGS process eliminates barriers associated with the use of the MinION and NGS in clinical laboratories.},
language = {en},
urldate = {2021-06-27},
journal = {Antiviral Research},
author = {Chorlton, Samuel D. and Ritchie, Gordon and Lawson, Tanya and McLachlan, Elizabeth and Romney, Marc G. and Matic, Nancy and Lowe, Christopher F.},
month = aug,
year = {2021},
keywords = {CMV, Sequencing, UL54, UL97},
pages = {105123},
}
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Methods Retrospective review of CMV AVDR testing requests for UL97 and UL54 at our laboratory from 2014 to 2019 was conducted. NGS was performed on the MinION and compared to Sanger sequencing performed at the national reference laboratory. Analysis of the sequences was completed with a novel cloud bioinformatics platform (BugSeq). Results Twenty patient samples previously characterized were included for study on the MinION. NGS captured all of the CMV AVDR mutations identified by Sanger, and identified additional mutations in UL97 and/or UL54 in 8/13 (62%) of the samples. An analysis of the depth of coverage at which we no longer detected minority single nucleotide variants (SNVs) detected in the original data was conducted, estimating a recall of 95% at 1800 fold coverage. Conclusion NGS utilizing MinION technology for the detection of CMV AVDR mutations identified additional minority variants in UL97 and UL54 as compared with Sanger sequencing. Through the application of a bioinformatics pipeline available online, our NGS process eliminates barriers associated with the use of the MinION and NGS in clinical laboratories.","language":"en","urldate":"2021-06-27","journal":"Antiviral Research","author":[{"propositions":[],"lastnames":["Chorlton"],"firstnames":["Samuel","D."],"suffixes":[]},{"propositions":[],"lastnames":["Ritchie"],"firstnames":["Gordon"],"suffixes":[]},{"propositions":[],"lastnames":["Lawson"],"firstnames":["Tanya"],"suffixes":[]},{"propositions":[],"lastnames":["McLachlan"],"firstnames":["Elizabeth"],"suffixes":[]},{"propositions":[],"lastnames":["Romney"],"firstnames":["Marc","G."],"suffixes":[]},{"propositions":[],"lastnames":["Matic"],"firstnames":["Nancy"],"suffixes":[]},{"propositions":[],"lastnames":["Lowe"],"firstnames":["Christopher","F."],"suffixes":[]}],"month":"August","year":"2021","keywords":"CMV, Sequencing, UL54, UL97","pages":"105123","bibtex":"@article{chorlton_next-generation_2021,\n\ttitle = {Next-generation sequencing for cytomegalovirus antiviral resistance genotyping in a clinical virology laboratory},\n\tvolume = {192},\n\tissn = {0166-3542},\n\turl = {https://samchorlton.com/papers/NGS_CMV_AVDR.pdf},\n\tdoi = {10.1016/j.antiviral.2021.105123},\n\tabstract = {Introduction\nThe identification of CMV antiviral drug resistance (AVDR) is a critical diagnostic test for immunocompromised patients with CMV infection and a failure of virologic response on optimal antiviral treatment. We developed a next-generation sequencing (NGS) assay for CMV AVDR testing and compared the AVDR mutations identified by NGS to Sanger sequencing.\nMethods\nRetrospective review of CMV AVDR testing requests for UL97 and UL54 at our laboratory from 2014 to 2019 was conducted. NGS was performed on the MinION and compared to Sanger sequencing performed at the national reference laboratory. Analysis of the sequences was completed with a novel cloud bioinformatics platform (BugSeq).\nResults\nTwenty patient samples previously characterized were included for study on the MinION. NGS captured all of the CMV AVDR mutations identified by Sanger, and identified additional mutations in UL97 and/or UL54 in 8/13 (62\\%) of the samples. An analysis of the depth of coverage at which we no longer detected minority single nucleotide variants (SNVs) detected in the original data was conducted, estimating a recall of 95\\% at 1800 fold coverage.\nConclusion\nNGS utilizing MinION technology for the detection of CMV AVDR mutations identified additional minority variants in UL97 and UL54 as compared with Sanger sequencing. Through the application of a bioinformatics pipeline available online, our NGS process eliminates barriers associated with the use of the MinION and NGS in clinical laboratories.},\n\tlanguage = {en},\n\turldate = {2021-06-27},\n\tjournal = {Antiviral Research},\n\tauthor = {Chorlton, Samuel D. and Ritchie, Gordon and Lawson, Tanya and McLachlan, Elizabeth and Romney, Marc G. and Matic, Nancy and Lowe, Christopher F.},\n\tmonth = aug,\n\tyear = {2021},\n\tkeywords = {CMV, Sequencing, UL54, UL97},\n\tpages = {105123},\n}\n\n","author_short":["Chorlton, S. 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