Establishment of performance parameters for the Qubit™ 1X dsDNA HS Assay for use in forensic genetics casework
Next generation sequencing is being more regularly used in forensic casework, particularly for post-mortem genetic analyses in sudden unexpected death cases. Accurate quantification of DNA and associated sequencing libraries is essential, however, no validation studies have been published on the rec...
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| Format: | Article |
| Language: | English |
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Elsevier
2025-07-01
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| Series: | Forensic Science International: Reports |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2665910725000015 |
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| author | Adele Lauren Kaleigh Naidoo Donna-Lee Pamela Martin Laura Jane Heathfield |
| author_facet | Adele Lauren Kaleigh Naidoo Donna-Lee Pamela Martin Laura Jane Heathfield |
| author_sort | Adele Lauren Kaleigh Naidoo |
| collection | DOAJ |
| description | Next generation sequencing is being more regularly used in forensic casework, particularly for post-mortem genetic analyses in sudden unexpected death cases. Accurate quantification of DNA and associated sequencing libraries is essential, however, no validation studies have been published on the recommended fluorometric quantification methods. The aim of this study was to optimise and internally validate the Qubit™ 1X dsDNA HS Assay Kit on the Qubit™ 4 Fluorometer as part of a molecular autopsy workflow for forensic applications. An optimised working solution equilibration period, sample incubation time and dilution medium were established. The optimised workflow was then used to assess the DNA concentration of control Lambda DNA, DNA extracted from forensic samples and sequencing libraries. The accuracy, precision, dynamic range, and sensitivity were established in accordance with ISO 17025 standards. All parameters met the manufacturer’s criteria of acceptance except for the precision of measurements for samples with DNA concentrations greater than or equal to 0.5 ng/µl, which were expected to have less than 1 % CV. The precision of the measurements (1.2–2.44 % CV), however, was deemed acceptable for our laboratory, as downstream DNA sequencing results surpassed quality thresholds. Additionally, precision between operators was poor. This study has advanced our progression towards implementing a molecular autopsy sequencing workflow in South Africa and provides the first performance parameters for the Qubit™ 1X dsDNA HS Assay Kit which may be useful for other laboratories. |
| format | Article |
| id | doaj-art-3b66148cbc714b4cb4dff505b2e837b6 |
| institution | Kabale University |
| issn | 2665-9107 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Forensic Science International: Reports |
| spelling | doaj-art-3b66148cbc714b4cb4dff505b2e837b62025-01-16T04:29:14ZengElsevierForensic Science International: Reports2665-91072025-07-0111100405Establishment of performance parameters for the Qubit™ 1X dsDNA HS Assay for use in forensic genetics caseworkAdele Lauren Kaleigh Naidoo0Donna-Lee Pamela Martin1Laura Jane Heathfield2Division of Forensic Medicine and Toxicology, Department of Pathology, Faculty of Health Sciences, University of Cape Town, South AfricaDivision of Forensic Medicine and Toxicology, Department of Pathology, Faculty of Health Sciences, University of Cape Town, South AfricaCorrespondence to: Division of Forensic Medicine and Toxicology, P.O Box 13914, Mowbray 7705, South Africa.; Division of Forensic Medicine and Toxicology, Department of Pathology, Faculty of Health Sciences, University of Cape Town, South AfricaNext generation sequencing is being more regularly used in forensic casework, particularly for post-mortem genetic analyses in sudden unexpected death cases. Accurate quantification of DNA and associated sequencing libraries is essential, however, no validation studies have been published on the recommended fluorometric quantification methods. The aim of this study was to optimise and internally validate the Qubit™ 1X dsDNA HS Assay Kit on the Qubit™ 4 Fluorometer as part of a molecular autopsy workflow for forensic applications. An optimised working solution equilibration period, sample incubation time and dilution medium were established. The optimised workflow was then used to assess the DNA concentration of control Lambda DNA, DNA extracted from forensic samples and sequencing libraries. The accuracy, precision, dynamic range, and sensitivity were established in accordance with ISO 17025 standards. All parameters met the manufacturer’s criteria of acceptance except for the precision of measurements for samples with DNA concentrations greater than or equal to 0.5 ng/µl, which were expected to have less than 1 % CV. The precision of the measurements (1.2–2.44 % CV), however, was deemed acceptable for our laboratory, as downstream DNA sequencing results surpassed quality thresholds. Additionally, precision between operators was poor. This study has advanced our progression towards implementing a molecular autopsy sequencing workflow in South Africa and provides the first performance parameters for the Qubit™ 1X dsDNA HS Assay Kit which may be useful for other laboratories.http://www.sciencedirect.com/science/article/pii/S2665910725000015Qubit™ 1X dsDNA HS AssayInternal validationISO17025NGS library quantificationMolecular autopsy |
| spellingShingle | Adele Lauren Kaleigh Naidoo Donna-Lee Pamela Martin Laura Jane Heathfield Establishment of performance parameters for the Qubit™ 1X dsDNA HS Assay for use in forensic genetics casework Forensic Science International: Reports Qubit™ 1X dsDNA HS Assay Internal validation ISO17025 NGS library quantification Molecular autopsy |
| title | Establishment of performance parameters for the Qubit™ 1X dsDNA HS Assay for use in forensic genetics casework |
| title_full | Establishment of performance parameters for the Qubit™ 1X dsDNA HS Assay for use in forensic genetics casework |
| title_fullStr | Establishment of performance parameters for the Qubit™ 1X dsDNA HS Assay for use in forensic genetics casework |
| title_full_unstemmed | Establishment of performance parameters for the Qubit™ 1X dsDNA HS Assay for use in forensic genetics casework |
| title_short | Establishment of performance parameters for the Qubit™ 1X dsDNA HS Assay for use in forensic genetics casework |
| title_sort | establishment of performance parameters for the qubit™ 1x dsdna hs assay for use in forensic genetics casework |
| topic | Qubit™ 1X dsDNA HS Assay Internal validation ISO17025 NGS library quantification Molecular autopsy |
| url | http://www.sciencedirect.com/science/article/pii/S2665910725000015 |
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