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Analytical Comparison of Methods for Extraction of Short Cell-Free DNA from Urine.

ABSTRACT: Urine cell-free DNA (cfDNA) is a valuable noninvasive biomarker for cancer mutation detection, infectious disease diagnosis (eg, tuberculosis), organ transplantation monitoring, and prenatal screening. Conventional silica DNA extraction does not efficiently capture urine cfDNA, which is dilute (ng/mL) and highly fragmented [30 to 100 nucleotides (nt)]. The clinical sensitivity of urine cfDNA detection increases with decreasing target length, motivating use of sample preparation methods designed for short fragments. We compared the analytical performance of two published protocols (Wizard resin/guanidinium thiocyanate and Q Sepharose), three commercial kits (Norgen, QIAamp, and MagMAX), and an in-house sequence-specific hybridization capture technique. Dependence on fragment length (25 to 150 nt), performance at low concentrations (10 copies/mL), tolerance to variable urine conditions, and susceptibility to PCR inhibition were characterized. Hybridization capture and Q Sepharose performed best overall (60% to 90% recovery), although Q Sepharose had reduced recovery (<10%) of the shortest 25-nt fragment. Wizard resin/guanidinium thiocyanate recovery was dependent on pH and background DNA concentration and was limited to <35%, even under optimal conditions. The Norgen kit led to consistent PCR inhibition but had high recovery of short fragments. The QIAamp and MagMAX kits had minimal recovery of fragments <150 and <80 nt, respectively. Urine cfDNA extraction methods differ widely in ability to capture short, dilute cfDNA in urine; using suboptimal methods may profoundly impair clinical results.

SUBMITTER: Oreskovic A

PROVIDER: S-EPMC6854475 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Analytical Comparison of Methods for Extraction of Short Cell-Free DNA from Urine.

Oreskovic Amy A Brault Norman D ND Panpradist Nuttada N Lai James J JJ Lutz Barry R BR

The Journal of molecular diagnostics : JMD 20190820 6

Urine cell-free DNA (cfDNA) is a valuable noninvasive biomarker for cancer mutation detection, infectious disease diagnosis (eg, tuberculosis), organ transplantation monitoring, and prenatal screening. Conventional silica DNA extraction does not efficiently capture urine cfDNA, which is dilute (ng/mL) and highly fragmented [30 to 100 nucleotides (nt)]. The clinical sensitivity of urine cfDNA detection increases with decreasing target length, motivating use of sample preparation methods designed ...[more]

PMID: 31442674

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Project description:In allograft monitoring of solid organ transplant recipients, liquid biopsy has emerged as a novel approach using quantification of donor-derived cell-free DNA (dd-cfDNA) in plasma. Despite early clinical implementation and analytical validation of techniques, direct comparisons of dd-cfDNA quantification methods are lacking. Furthermore, data on dd-cfDNA in urine is scarce and high-throughput sequencing-based methods so far have not leveraged unique molecular identifiers (UMIs) for absolute dd-cfDNA quantification. Different dd-cfDNA quantification approaches were compared in urine and plasma of kidney and liver recipients: A) Droplet digital PCR (ddPCR) using allele-specific detection of seven common HLA-DRB1 alleles and the Y chromosome; B) high-throughput sequencing (HTS) using a custom QIAseq DNA panel targeting 121 common polymorphisms; and C) a commercial dd-cfDNA quantification method (AlloSeq® cfDNA, CareDx). Dd-cfDNA was quantified as %dd-cfDNA, and for ddPCR and HTS using UMIs additionally as donor copies. In addition, relative and absolute dd-cfDNA levels in urine and plasma were compared in clinically stable recipients. The HTS method presented here showed a strong correlation of the %dd-cfDNA with ddPCR (R 2 = 0.98) and AlloSeq® cfDNA (R 2 = 0.99) displaying only minimal to no proportional bias. Absolute dd-cfDNA copies also correlated strongly (τ = 0.78) between HTS with UMI and ddPCR albeit with substantial proportional bias (slope: 0.25; 95%-CI: 0.19-0.26). Among 30 stable kidney transplant recipients, the median %dd-cfDNA in urine was 39.5% (interquartile range, IQR: 21.8-58.5%) with 36.6 copies/μmol urinary creatinine (IQR: 18.4-109) and 0.19% (IQR: 0.01-0.43%) with 5.0 copies/ml (IQR: 1.8-12.9) in plasma without any correlation between body fluids. The median %dd-cfDNA in plasma from eight stable liver recipients was 2.2% (IQR: 0.72-4.1%) with 120 copies/ml (IQR: 85.0-138) while the median dd-cfDNA copies/ml was below 0.1 in urine. This first head-to-head comparison of methods for absolute and relative quantification of dd-cfDNA in urine and plasma supports a method-independent %dd-cfDNA cutoff and indicates the suitability of the presented HTS method for absolute dd-cfDNA quantification using UMIs. To evaluate the utility of dd-cfDNA in urine for allograft surveillance, absolute levels instead of relative amounts will most likely be required given the extensive variability of %dd-cfDNA in stable kidney recipients.

Project description:BackgroundTo reach non-participants, reluctant to undergo clinician-based cervical cancer screening and vaginal self-sampling, urine collection for high-risk human papillomavirus detection (hrHPV) may be valuable. Using two hrHPV DNA assays, we evaluated the concordance of hrHPV positivity in urine samples in comparison with vaginal self-samples and cervical cytology samples taken by the general practitioner (GP). We also studied women's acceptance of urine collection and preferences towards the different sampling procedures.MethodsOne hundred fifty paired self-collected urine and vaginal samples and GP-collected cervical cytology samples were obtained from 30 to 59-year-old women diagnosed with ASC-US within the Danish cervical cancer screening program. After undergoing cervical cytology at the GP, the women collected first-void urine and vaginal samples at home and completed a questionnaire. Each sample was hrHPV DNA tested by the GENOMICA CLART® and COBAS® 4800 assays. Concordance in hrHPV detection between sample types was determined using Kappa (k) statistics. Sensitivity and specificity of hrHPV detection in urine was calculated using cervical sampling as reference.ResultsWith the COBAS assay, urine showed good concordance to the vaginal (k = 0.66) self-samples and cervical samples (k = 0.66) for hrHPV detection. The corresponding concordance was moderate (k = 0.59 and k = 0.47) using CLART. Compared to cervical sampling, urinary hrHPV detection had a sensitivity of 63.9% and a specificity of 96.5% using COBAS; compared with 51.6 and 92.4% for CLART. Invalid hrHPV test rates were 1.8% for COBAS and 26.9% for CLART. Urine collection was well-accepted and 42.3% of the women ranked it as the most preferred future screening procedure.ConclusionsUrine collection provides a well-accepted screening option. With COBAS, higher concordance between urine and vaginal self-sampling and cervical sampling for hrHPV detection was found compared to CLART. Urinary hrHPV detection with COBAS is feasible, but its accuracy may need to be improved before urine collection at home can be offered to non-participants reluctant to both cervical sampling and vaginal self-sampling.

Dataset Information

Analytical Comparison of Methods for Extraction of Short Cell-Free DNA from Urine.

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Analytical Comparison of Methods for Extraction of Short Cell-Free DNA from Urine.

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