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

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. 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. CME Accreditation Statement: This activity ("JMD 2019 CME Program in Molecular Diagnostics") has been planned and implemented in accordance with the accreditation requirements and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint providership of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity ("JMD 2019 CME Program in Molecular Diagnostics") for a maximum of 18.0 AMA PRA Category 1 Credit(s)™. Physicians should claim only credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. CME Accreditation Statement: This activity ("JMD 2019 CME Program in Molecular Diagnostics") has been planned and implemented in accordance with the accreditation requirements and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint providership of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity ("JMD 2019 CME Program in Molecular Diagnostics") for a maximum of 18.0 AMA PRA Category 1 Credit(s)™. Physicians should claim only credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Urine cell-free DNA (cfDNA) is an emerging noninvasive biomarker for cancer mutation detection,1Botezatu I. Serdyuk O. Potapova G. Shelepov V. Alechina R. Molyaka Y. Ananév V. Bazin I. Garin A. Narimanov M. Knysh V. Melkonyan H. Umansky S. Lichtenstein A. Genetic analysis of DNA excreted in urine: a new approach for detecting specific genomic DNA sequences from cells dying in an organism.Clin Chem. 2000; 46: 1078-1084Crossref PubMed Scopus (290) Google Scholar, 2Reckamp K.L. 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Potapova G. Shelepov V. Alechina R. Molyaka Y. Ananév V. Bazin I. Garin A. Narimanov M. Knysh V. Melkonyan H. Umansky S. Lichtenstein A. Genetic analysis of DNA excreted in urine: a new approach for detecting specific genomic DNA sequences from cells dying in an organism.Clin Chem. 2000; 46: 1078-1084Crossref PubMed Scopus (290) Google Scholar This subset of urine cfDNA, derived from circulating cfDNA, is known as transrenal DNA, but cfDNA can also be generated directly in urine from cells shed along the urinary tract. To maximize the clinical sensitivity and reproducibility of urine cfDNA analysis, extraction methods capable of efficiently capturing short, dilute DNA fragments are essential. Although plasma cfDNA is primarily nucleosomal, with a peak length of 160 to 167 nucleotides (nt),12Burnham P. Kim M.S. Agbor-Enoh S. Luikart H. Valantine H.A. Khush K.K. De Vlaminck I. Single-stranded DNA library preparation uncovers the origin and diversity of ultrashort cell-free DNA in plasma.Sci Rep. 2016; 6: 27859Crossref PubMed Scopus (120) Google Scholar, 13Underhill H.R. Kitzman J.O. Hellwig S. Welker N.C. Daza R. Baker D.N. Gligorich K.M. Rostomily R.C. Bronner M.P. Shendure J. Fragment length of circulating tumor DNA.PLoS Genet. 2016; 12: e1006162Crossref PubMed Scopus (393) Google Scholar, 14Yu S.C.Y. Lee S.W.Y. Jiang P. Leung T.Y. Chan K.C.A. Chiu R.W.K. Lo Y.M.D. High-resolution profiling of fetal DNA clearance from maternal plasma by massively parallel sequencing.Clin Chem. 2013; 59: 1228-1237Crossref PubMed Scopus (166) Google Scholar urine cfDNA is more fragmented. The upper length limit of the transrenal fraction of urine cfDNA is defined by glomerular filtration, and all urine cfDNA fragments are quickly degraded further in urine.15Yao W. Mei C. Nan X. Hui L. Evaluation and comparison of in vitro degradation kinetics of DNA in serum, urine and saliva: a qualitative study.Gene. 2016; 590: 142-148Crossref PubMed Scopus (121) Google Scholar Determining the true length distribution of urine cfDNA is challenging because both extraction and library preparation methods may underestimate the presence of shorter fragments, but most fragments are expected to be <100 nt.11Tsui N.B.Y. Jiang P. Chow K.C.K. Su X. Leung T.Y. Sun H. Chan K.C.A. Chiu R.W.K. Lo Y.M.D. High resolution size analysis of fetal DNA in the urine of pregnant women by paired-end massively parallel sequencing.PLoS One. 2012; 7: e48319Crossref PubMed Scopus (69) Google Scholar, 14Yu S.C.Y. Lee S.W.Y. Jiang P. Leung T.Y. Chan K.C.A. Chiu R.W.K. Lo Y.M.D. High-resolution profiling of fetal DNA clearance from maternal plasma by massively parallel sequencing.Clin Chem. 2013; 59: 1228-1237Crossref PubMed Scopus (166) Google Scholar, 16Burnham P. Dadhania D. Heyang M. Chen F. Westblade L.F. Suthanthiran M. Lee J.R. De Vlaminck I. Urinary cell-free DNA is a versatile analyte for monitoring infections of the urinary tract.Nat Commun. 2018; 9: 2412Crossref PubMed Scopus (91) Google Scholar Peak fragment length varies across patients, but may be as low as 30 to 60 nt.11Tsui N.B.Y. Jiang P. Chow K.C.K. Su X. Leung T.Y. Sun H. Chan K.C.A. Chiu R.W.K. Lo Y.M.D. High resolution size analysis of fetal DNA in the urine of pregnant women by paired-end massively parallel sequencing.PLoS One. 2012; 7: e48319Crossref PubMed Scopus (69) Google Scholar, 14Yu S.C.Y. Lee S.W.Y. Jiang P. Leung T.Y. Chan K.C.A. Chiu R.W.K. Lo Y.M.D. High-resolution profiling of fetal DNA clearance from maternal plasma by massively parallel sequencing.Clin Chem. 2013; 59: 1228-1237Crossref PubMed Scopus (166) Google Scholar, 16Burnham P. Dadhania D. Heyang M. Chen F. Westblade L.F. Suthanthiran M. Lee J.R. De Vlaminck I. Urinary cell-free DNA is a versatile analyte for monitoring infections of the urinary tract.Nat Commun. 2018; 9: 2412Crossref PubMed Scopus (91) Google Scholar Because of the extensive fragmentation of urine cfDNA, the diagnostic clinical sensitivity of urine cfDNA detection increases with decreasing target length. Maximizing sensitivity by targeting shorter fragments is especially critical because urine cfDNA is also dilute, with total concentrations ranging from <1 to 200 ng/mL1Botezatu I. Serdyuk O. Potapova G. Shelepov V. Alechina R. Molyaka Y. Ananév V. Bazin I. Garin A. Narimanov M. Knysh V. Melkonyan H. Umansky S. Lichtenstein A. Genetic analysis of DNA excreted in urine: a new approach for detecting specific genomic DNA sequences from cells dying in an organism.Clin Chem. 2000; 46: 1078-1084Crossref PubMed Scopus (290) Google Scholar, 17Su Y.-H. Wang M. Brenner D.E. Ng A. Melkonyan H. Umansky S. Syngal S. Block T.M. Human urine contains small, 150 to 250 nucleotide-sized, soluble DNA derived from the circulation and may be useful in the detection of colorectal cancer.J Mol Diagn. 2004; 6: 101-107Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 18Streleckiene G. Reid H.M. Arnold N. Bauerschlag D. Forster M. Quantifying cell free DNA in urine: comparison between commercial kits, impact of gender and inter-individual variation.Biotechniques. 2018; 64: 225-230Crossref PubMed Scopus (10) Google Scholar and copy numbers of specific targets much lower. In a study detecting fetal cfDNA in maternal urine, decreasing PCR amplicon length from 65 to 39 nt increased clinical sensitivity from 25% to 75%. A further decrease to 25 nt was required before achieving 100% detection.10Shekhtman E.M. Anne K. Melkonyan H.S. Robbins D.J. Warsof S.L. Umansky S.R. Optimization of transrenal DNA analysis: detection of fetal DNA in maternal urine.Clin Chem. 2009; 55: 723-729Crossref PubMed Scopus (52) Google Scholar This effect may be even more pronounced for bacterial, viral, and mitochondrial cfDNA, which are not protected by histones and are, therefore, more degraded than human genomic cfDNA.12Burnham P. Kim M.S. Agbor-Enoh S. Luikart H. Valantine H.A. Khush K.K. De Vlaminck I. Single-stranded DNA library preparation uncovers the origin and diversity of ultrashort cell-free DNA in plasma.Sci Rep. 2016; 6: 27859Crossref PubMed Scopus (120) Google Scholar, 15Yao W. Mei C. Nan X. Hui L. Evaluation and comparison of in vitro degradation kinetics of DNA in serum, urine and saliva: a qualitative study.Gene. 2016; 590: 142-148Crossref PubMed Scopus (121) Google Scholar, 16Burnham P. Dadhania D. Heyang M. Chen F. Westblade L.F. Suthanthiran M. Lee J.R. De Vlaminck I. Urinary cell-free DNA is a versatile analyte for monitoring infections of the urinary tract.Nat Commun. 2018; 9: 2412Crossref PubMed Scopus (91) Google Scholar For tuberculosis urine cfDNA, a modest 10-nt decrease in amplicon length (49 to 39 nt) led to 5- to 10-fold improvement in detected concentration.19Melkonyan H.S. Feaver W.J. Meyer E. Scheinker V. Shekhtman E.M. Xin Z. Umansky S.R. Transrenal nucleic acids: from proof of principle to clinical tests.Ann N Y Acad Sci. 2008; 1137: 73-81Crossref PubMed Scopus (88) Google Scholar Critically, the ability to target shorter cfDNA fragments lies not only in decreasing amplicon length, but also in design and selection of sample preparation methods capable of capturing and concentrating the short, dilute fragments that constitute the bulk of urine cfDNA. Unfortunately, conventional extraction methods for cell-associated DNA or even plasma cfDNA are not suitable for urine cfDNA because they are not designed for short fragments. The Boom method, commonly used for both research and clinical work, adsorbs DNA to silica under chaotropic conditions.20Boom R. Sol C.J. Salimans M.M. Jansen C.L. Wertheim-van Dillen P.M. van der Noordaa J. Rapid and simple method for purification of nucleic acids.J Clin Microbiol. 1990; 28: 495-503Crossref PubMed Google Scholar The key driving forces of silica adsorption are hydrophobic interactions due to dehydration of silica and DNA surfaces and hydrogen bonding between silica and the DNA backbone, both of which depend on DNA length.21Melzak K.A. Sherwood C.S. Turner R.F.B. Haynes C.A. Driving forces for DNA adsorption to silica in perchlorate solutions.J Colloid Interface Sci. 1996; 181: 635-644Crossref Scopus (324) Google Scholar Consequently, silica adsorption is less effective at purifying short fragments, with recovery generally decreasing below 50 to 100 nt. Silica adsorption also requires relatively high DNA concentrations for optimal performance because a fraction of DNA may remain irretrievably bound to the silica surface.22Katevatis C. Fan A. Klapperich C.M. Low concentration DNA extraction and recovery using a silica solid phase.PLoS One. 2017; 12: e0176848Crossref PubMed Scopus (56) Google Scholar, 23Vandeventer P.E. Lin J.S. Zwang T.J. Nadim A. Johal M.S. Niemz A. Multiphasic DNA adsorption to silica surfaces under varying buffer, pH, and ionic strength conditions.J Phys Chem B. 2012; 116: 5661-5670Crossref PubMed Scopus (81) Google Scholar This loss is trivial in most samples, but for low-concentration samples, like urine cfDNA, it may make up a significant portion of the input. With these limitations in mind, an ideal urine cfDNA extraction method would enable high recovery of short DNA from dilute solutions. Despite the great clinical promise of urine cfDNA as an easy-to-access sample, there has been little quantitative comparison of approaches taken to improve recovery of urine cfDNA. A recent review emphasized the lack of standardization in sample preparation methods, including DNA extraction, as a key limitation in the development of urine cfDNA assays.24Fernández-Carballo B.L. Broger T. Wyss R. Banaei N. Denkinger C.M. Towards the development of a cfDNA-based in-vitro diagnostic test for infectious diseases: a review of evidence for tuberculosis.Clin Microbiol. 2019; 57: e01234-18Crossref Scopus (35) Google Scholar Previous studies have compared clinical detection rates10Shekhtman E.M. Anne K. Melkonyan H.S. Robbins D.J. Warsof S.L. Umansky S.R. Optimization of transrenal DNA analysis: detection of fetal DNA in maternal urine.Clin Chem. 2009; 55: 723-729Crossref PubMed Scopus (52) Google Scholar, 19Melkonyan H.S. Feaver W.J. Meyer E. Scheinker V. Shekhtman E.M. Xin Z. Umansky S.R. Transrenal nucleic acids: from proof of principle to clinical tests.Ann N Y Acad Sci. 2008; 1137: 73-81Crossref PubMed Scopus (88) Google Scholar and total cfDNA recovery18Streleckiene G. Reid H.M. Arnold N. Bauerschlag D. Forster M. Quantifying cell free DNA in urine: comparison between commercial kits, impact of gender and inter-individual variation.Biotechniques. 2018; 64: 225-230Crossref PubMed Scopus (10) Google Scholar, 25El Bali L. Diman A. Bernard A. Roosens N.H.C. De Keersmaecker S.C.J. Comparative study of seven commercial kits for human DNA extraction from urine samples suitable for DNA biomarker-based public health studies.J Biomol Tech. 2014; 25: 96-110PubMed Google Scholar of a limited set of extraction methods, but no studies have investigated analytical performance using spiked samples. Herein, two published urine cfDNA extraction protocols [Wizard resin/guanidinium thiocyanate (Wizard/GuSCN) and Q Sepharose], three commercial kits (Norgen, QIAamp, and MagMAX), and a sequence-specific hybridization capture technique, developed in our laboratory, were analytically compared. The Wizard/GuSCN method uses high concentrations (>3 mol/L) of chaotropic GuSCN to adsorb DNA to Wizard silica resin. This approach was used to originally demonstrate the presence of cfDNA in urine1Botezatu I. Serdyuk O. Potapova G. Shelepov V. Alechina R. Molyaka Y. Ananév V. Bazin I. Garin A. Narimanov M. Knysh V. Melkonyan H. Umansky S. Lichtenstein A. Genetic analysis of DNA excreted in urine: a new approach for detecting specific genomic DNA sequences from cells dying in an organism.Clin Chem. 2000; 46: 1078-1084Crossref PubMed Scopus (290) Google Scholar and has since been widely applied, most frequently for detecting tuberculosis26Cannas A. Goletti D. Girardi E. Chiacchio T. Calvo L. Cuzzi G. Piacentini M. Melkonyan H. Umansky S.R. Lauria F.N. Ippolito G. Tomei L.D. Mycobacterium tuberculosis DNA detection in soluble fraction of urine from pulmonary tuberculosis patients.Int J Tuberc Lung Dis. 2008; 12: 146-151PubMed Google Scholar and fetal11Tsui N.B.Y. Jiang P. Chow K.C.K. Su X. Leung T.Y. Sun H. Chan K.C.A. Chiu R.W.K. Lo Y.M.D. High resolution size analysis of fetal DNA in the urine of pregnant women by paired-end massively parallel sequencing.PLoS One. 2012; 7: e48319Crossref PubMed Scopus (69) Google Scholar cfDNA. The Q Sepharose method uses a quaternary ammonium anion exchange resin to preconcentrate DNA before desalting on a silica spin column. It improves recovery of short urine cfDNA fragments compared with Wizard/GuSCN10Shekhtman E.M. Anne K. Melkonyan H.S. Robbins D.J. Warsof S.L. Umansky S.R. Optimization of transrenal DNA analysis: detection of fetal DNA in maternal urine.Clin Chem. 2009; 55: 723-729Crossref PubMed Scopus (52) Google Scholar and has often been used to detect tumor cfDNA mutations for cancer diagnosis, monitoring, and prognosis.2Reckamp K.L. Melnikova V.O. Karlovich C. Sequist L.V. Camidge D.R. Wakelee H. Perol M. Oxnard G.R. Kosco K. Croucher P. Samuelsz E. Vibat C.R. Guerrero S. Geis J. Berz D. Mann E. Matheny S. Rolfe L. Raponi M. Erlander M.G. Gadgeel S. A highly sensitive and quantitative test platform for detection of NSCLC EGFR mutations in urine and plasma.J Thorac Oncol. 2016; 11: 1690-1700Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 3Fujii T. Barzi A. Sartore-Bianchi A. Cassingena A. Siravegna G. Karp D.D. Piha-Paul S.A. Subbiah V. Tsimberidou A.M. Huang H.J. Veronese S. Di Nicolantonio F. Pingle S. Vibat C.R.T. Hancock S. Berz D. Melnikova V.O. Erlander M.G. Luthra R. Kopetz E.S. Meric-Bernstam F. Siena S. Lenz H.J. 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Factors that influence quality and yield of circulating-free DNA: a systematic review of the methodology literature.Heliyon. 2018; 4: e00699Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar but is not commonly used for urine cfDNA. The Thermo Fisher Scientific (Waltham, MA) MagMAX Cell-Free DNA Isolation Kit uses Dynabeads MyOne Silane to maximize binding kinetics and capacity but is intended primarily for plasma cfDNA. It was included as a reference method to represent best-case silica adsorption without modifications specifically for urine cfDNA, although it has been used previously in urine.28Lee D.H. Yoon H. Park S. Kim J.S. Ahn Y.H. Kwon K. Lee D. Kim K.H. Urinary exosomal and cell-free DNA detects somatic mutation and copy number alteration in urothelial carcinoma of bladder.Sci Rep. 2018; 8: 14707Crossref PubMed Scopus (39) Google Scholar To enable high-efficiency purification of short fragments, our laboratory has developed a hybridization capture method for urine cfDNA using a biotinylated sequence-specific probe and streptavidin-coated magnetic beads. Hybridization is commonly used for targeted enrichment of sequencing libraries but has been less frequently used as a sample preparation method for capturing target sequences directly from raw samples. Hybridization capture with magnetic beads has been used previously to enrich pathogen DNA and mRNA directly from sputum,29Mangiapan G. Vokurka M. Schouls L. Cadranel J. Lecossier D. Van Embden J. Hance A.J. Sequence capture-PCR improves detection of mycobacterial DNA in clinical specimens.J Clin Microbiol. 1996; 34: 1209-1215Crossref PubMed Google Scholar, 30Reed J.L. Basu D. 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Microfluidic platform for isolating nucleic acid targets using sequence specific hybridization.Biomicrofluidics. 2013; 7: 44107Crossref PubMed Scopus (17) Google Scholar and lateral flow38Rohrman B. Richards-Kortum R. Inhibition of recombinase polymerase amplification by background DNA: a lateral flow-based method for enriching target DNA.Anal Chem. 2015; 87: 1963-1967Crossref PubMed Scopus (77) Google Scholar formats. In previous implementations, hybridization capture was used primarily to remove excess nontarget DNA, which can inhibit amplification.30Reed J.L. Basu D. Butzler M.A. McFall S.M. XtracTB Assay, a Mycobacterium tuberculosis molecular screening test with sensitivity approaching culture.Sci Rep. 2017; 7: 3653Crossref PubMed Scopus (16) Google Scholar, 38Rohrman B. Richards-Kortum R. Inhibition of recombinase polymerase amplification by background DNA: a lateral flow-based method for enriching target DNA.Anal Chem. 2015; 87: 1963-1967Crossref PubMed Scopus (77) Google Scholar In the case of urine cfDNA, hybridization's ability to sensitively capture short fragments, regardless of length and concentration, was instead leveraged. To our knowledge, hybridization capture has not been used previously to target urine cfDNA. For each extraction method, the dependence on DNA fragment length, performance at low DNA concentrations, tolerance to variable urine conditions, and susceptibility to PCR inhibition were characterized. The results of this work will help guide selection and optimization of DNA extraction methods for urine cfDNA analysis. Careful design of sample preparation methods should lead to increased clinical sensitivity and reprodu