genomewide bisulfite sequencing reveals the origin and time dependent fragmentation of urinary cfdna

Accepted Manuscript Genomewide bisulfite sequencing reveals the origin and timedependent fragmentation of urinary cfDNA Timothy H.T Cheng, Peiyong Jiang, Jacqueline C.W Tam, Xiao Sun, Wing-Shan Lee, Stephanie C.Y Yu, Jeremy Y.C Teoh, Peter K.F Chiu, Chi-Fai Ng, Kai-Ming Chow, Cheuk-Chun Szeto, K.C Allen Chan, Rossa W.K Chiu, Y.M Dennis Lo PII: DOI: Reference: S0009-9120(16)30718-4 doi: 10.1016/j.clinbiochem.2017.02.017 CLB 9483 To appear in: Clinical Biochemistry Received date: Revised date: Accepted date: 23 December 2016 February 2017 21 February 2017 Please cite this article as: Timothy H.T Cheng, Peiyong Jiang, Jacqueline C.W Tam, Xiao Sun, Wing-Shan Lee, Stephanie C.Y Yu, Jeremy Y.C Teoh, Peter K.F Chiu, Chi-Fai Ng, Kai-Ming Chow, Cheuk-Chun Szeto, K.C Allen Chan, Rossa W.K Chiu, Y.M Dennis Lo , Genomewide bisulfite sequencing reveals the origin and time-dependent fragmentation of urinary cfDNA The address for the corresponding author was captured as affiliation for all authors Please check if appropriate Clb(2016), doi: 10.1016/j.clinbiochem.2017.02.017 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain ACCEPTED MANUSCRIPT Genomewide bisulfite sequencing reveals the origin and timedependent fragmentation of urinary cfDNA Timothy H.T Cheng1,2, Peiyong Jiang1,2, Jacqueline C.W Tam1,2, Xiao Sun1,2, Wing-Shan SC RI PT Lee1,2, Stephanie C.Y Yu1,2, Jeremy Y.C Teoh3, Peter K.F Chiu3, Chi-Fai Ng3, Kai-Ming Chow4, Cheuk-Chun Szeto4, K.C Allen Chan1,2, Rossa W.K Chiu1,2 and Y.M Dennis Lo1,2* New Territories, Hong Kong SAR, China NU Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Shatin, MA Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR, China ED SH Ho Urology Centre, Department of Surgery, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR, China PT Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Prince CE of Wales Hospital, Shatin, New Territories, Hong Kong SAR, China AC *To whom correspondence may be addressed: Y.M Dennis Lo; Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, 30–32 Ngan Shing Street, Shatin, New Territories, Hong Kong SAR, China; Tel +852 37636001; Fax +852 26365090; E-mail loym@cuhk.edu.hk ACCEPTED MANUSCRIPT Abstract Urinary cell-free (cf) DNA holds great potential as a completely noninvasive form of liquid biopsy Knowledge of the composition of cfDNA by tissue of origin is useful for guiding its clinical uses We conducted a global survey of urinary cfDNA composition using SC RI PT genomewide bisulfite sequencing While previous studies focused on detecting cfDNA from a single source at a time, genomewide tissue specific methylation signatures allow us to simultaneously deduce the proportional contribution from each contributing tissue The proportional contributions derived from methylation deconvolution are highly correlated NU with those calculated using allograft-derived donor-specific genetic markers in the urine of hematopoetic stem cell and renal transplant recipients We found a large variation of MA proportional contributions from different tissues We then assessed if cfDNA undergoes time-dependent fragmentation in urine by conducting in vitro incubation experiments In ED vitro incubation at 37 °C showed that urinary cfDNA concentration decreased under first order kinetics with a half-life of 2.6 to 5.1 hours This is reflected in parallel by a decrease PT in the proportion of long fragments and increase in amplitude of 10 bp periodicity seen in CE the cfDNA size profile This global survey of urinary cfDNA has deepened our understanding of the composition, degradation and variation of cfDNA in the urinary tract AC and has laid a foundation for the use of genomewide urinary cfDNA sequencing as a molecular diagnostics tool Keywords: urinary cell-free DNA, liquid biopsy, genomewide bisulfite sequencing ACCEPTED MANUSCRIPT Introduction Short fragments of extracellular DNA found in human body fluids are released during apoptosis and necrosis from dying cells (1) Analyses of cell-free (cf) DNA circulating in plasma originating from the fetus (2), tumor cells (3) and transplant allograft (4) have SC RI PT enabled the development of noninvasive prenatal testing (5), ‘liquid biopsy’ for assessing tumors (6,7), and the monitoring of the clinical status of transplanted organs (8) Urine analysis is truly noninvasive and understanding the origin of urinary cfDNA is useful NU for guiding its clinical use as a form of ‘liquid biopsy’ DNA isolated from the cell-free supernatant of urine can be broadly categorized as arising from the pre-renal, renal and MA post-renal systems Using blood transfusion (9), pregnancy (9–11), hematopoietic stem cell transplantation (12), non-urologic malignancies (13,14), renal transplantation (15), and ED bladder cancer (16,17) as model systems, a number of groups have demonstrated that a proportion of urinary cfDNA is derived from the systemic circulation, the kidney and from CE PT the post-renal urothelium Previous studies typically focus on detecting cfDNA from a single source of interest at a AC time, and there is a large variation in the quantity of urinary cfDNA derived from a particular source The proportional contribution of each tissue source to the total urinary cfDNA is unknown, and in some studies, the concentration of cfDNA from the source of interest is extremely low, or even undetectable (15,16,18) CpG site methylation is an important form of epigenetic regulation and methylation signatures can be identified for different tissues (19,20) and cell types (21) We have recently demonstrated that the proportional contribution of cfDNA from different tissues ACCEPTED MANUSCRIPT can be ascertained using genomewide bisulfite sequencing and deconvolution analyses of the sequencing data (22,23) Here we aim to conduct a global survey of the composition of urinary cfDNA by employing methylation deconvolution to simultaneously infer the proportional contribution of different SC RI PT tissues to urinary cfDNA We then conducted in vitro incubation experiments to assess if cfDNA fragments undergo fragmentation in a time-dependent manner in urine Materials and Methods NU Sample collection and processing MA This study was approved by the Joint Chinese University of Hong Kong – Hospital Authority New Territories East Cluster Clinical Research Ethics Committee All study subjects were recruited from the Prince of Wales Hospital with informed consent, and all PT ED samples were collected from January 2014 to March 2016 26 urine samples were collected from 11 renal transplant patients and five urine samples CE were collected from two hematopoietic stem cell transplant (HSCT) patients Nine clinically AC stable renal transplant patients were selected based on availability of fresh frozen kidney biopsy tissue or donor buffy coat samples for genotyping These stable renal transplant patients had static plasma creatinine levels in successive follow-ups and were not undergoing acute rejection We also collected multiple urine samples from two transplant patients from day to day 70 post-transplant We aimed to collect urine from a range of plasma creatinine levels to see if we could observe a range of variation of contribution of cfDNA from the kidney Paired kidney pelvis urine (via percutaneous nephrostomy) and voided urine were collected from a patient with a large 2cm renal stone ACCEPTED MANUSCRIPT Urine samples were collected during the morning clinic, or the morning before surgery, with early morning urine samples being avoided if possible 30-50 mL of urine was collected in plain sterile bottles, stored at °C and processed within one hour of collection as previously described(12,24) The cell free portion of the urine was isolated by SC RI PT centrifugation and filtering of the supernatant (Supplementary Materials and Methods) Library preparation, bisulfite conversion and massively parallel DNA sequencing DNA libraries were prepared with up to 500 ng of urinary cfDNA using the KAPA HTP Library Preparation Kit (Kapa Biosystems) according to the manufacturer’s instructions (7) NU Bisulfite and non-bisulfite DNA sequencing were performed as previously described (25,26), using an Illumina HiSeq 2500 sequencer using the 75 bp paired end mode After MA base calling and quality control, the data were then processed by the methylation data analysis pipeline Methy-Pipe (27) See Supplementary Materials and Methods for PT ED additional details CE Results Identification of differentially methylated regions for urinary cfDNA tissue mapping AC We hypothesized that blood cells, the kidney and the urothelium were the major contributors, respectively, for the pre-renal, renal and post-renal release of cfDNA into urine Around 80% of the cfDNA in plasma is from hematopoietic cells (28) and thus if a significant amount of plasma cfDNA is able to be filtered through the kidney into the urine, these DNA fragments would likely bear characteristics of the hematopoietic cells We aimed to characterize the methylome of blood cells (neutrophils, T-cells and B-cells), the kidney and the urothelium in order to identify methylation signatures that could help us ACCEPTED MANUSCRIPT differentiate between these tissues We made use of publicly available whole genome bisulfite sequencing data for blood cells (Human Epigenome Atlas, www.genboree.org/epigenomeatlas/index.rhtml, (29)) and we obtained kidney and urothelial tissues from patients undergoing renal transplantation or urologic surgery, in order to perform whole genome bisulfite sequencing to 35 – 40X haploid genome SC RI PT coverage (see Supplementary Materials and Methods) Autosomal CpG islands and shores were subdivided into non-overlapping 500 bp units and the methylation density of each unit was determined for each reference tissue We identified 19,418 differentially methylated regions (DMRs) across the genome to be used NU as methylation markers as previously described ((22), Supplementary Fig 1) 3,549 DMRs MA were selected because they showed a grossly different methylation density (z-score >3) in one tissue compared with the other tissues A further 15,869 DMRs were selected ED because they exhibited highly variable methylation densities across different tissue types PT (variation in methylation density >20% and coefficient of variation > 0.25) Urinary cfDNA was sequenced after bisulfite treatment and the methylation patterns CE observed in cfDNA fragments at the DMRs were compared with the methylation signatures AC in the five reference tissues, and using the methylation deconvolution algorithm as previously described (22) We then inferred the proportional contributions of blood cells (neutrophils, B-cells, T-cells), kidney and urothelium Methylation deconvolution in hematopoietic stem cell and renal transplant patients and validation using donor-specific genotypes We ascertained donor and recipient germline genotype information using the Illumina OMNI 2M SNP arrays for HSCT and renal transplant patients We collected 31 urine ACCEPTED MANUSCRIPT samples for bisulfite sequencing The global methylation density ranged from 61.1% to 73.5% in these samples We obtained an average of 80 million uniquely mapped reads for each sample, and the identification of fragments harboring donor and recipient-specific SNPs allowed the accurate calculation of the proportion of cfDNA fragments from the donor tissues (See Supplementary Table for sequencing coverage for each sample) SC RI PT The proportional contributions of the donor tissues determined by methylation deconvolution and the proportions determined using donor-specific genotypes were highly correlated (R2=0.97, Fig 1) NU Variation in the proportional contribution from each tissue and concentration of kidney-derived cfDNA MA These results demonstrated the ability of methylation deconvolution to determine the proportional contribution of different tissues into urinary cfDNA over a good dynamic ED range Using donor-specific SNPs, the proportion of donor hematopoietic cell contribution to urinary cfDNA varied from 6-78%, and the proportion of donor kidney contribution varied PT from 1-94% The full urinary cfDNA methylation deconvolution results for the 31 transplant CE urine samples are listed in Table These results demonstrated that the contributions of blood cells, kidney and urothelium were highly variable between different samples The AC proportional contribution of each of these tissues can be as low as 0%, and can rise up to 93%, 100% and 64% for blood cells, kidney and urothelium, respectively Across the 31 urine samples the median and interquartile ranges of the proportional contributions measured using methylation deconvolution for blood cells, kidney and urothelium were 52% (0-84%), 32% (7-100%) and 5% (0-12%), respectively We have previously demonstrated that a grossly elevated concentration of urinary cfDNA originating from the kidney can be detected during an episode of acute rejection, and the ACCEPTED MANUSCRIPT concentration of kidney-derived cfDNA reduced rapidly with treatment (15) In our current study, the total concentration of urinary cfDNA in kidney transplant patients varied from 559 to 25,710 GE/ml urine The kidney-derived cfDNA concentrations calculated from SNP alleles varied from 65 to 3,971 GE/ml urine (8-923 GE/ml/mmol Cr after adjustment with urine creatinine levels) In our series of stable renal transplant patients, and patients SC RI PT with normalizing plasma creatinine levels in the post-transplant period, the proportion of kidney contribution to the total urinary cfDNA, and the kidney-derived cfDNA concentration (with or without creatinine adjustment) did not correlate plasma creatinine levels (Supplementary Fig 2) However, in two patients that we monitored serially in the acute post-transplant period, we observed a decreasing trend in the fraction of donor kidney MA NU contribution but not the total kidney-derived cfDNA concentration (Supplementary Table 2) ED Time-dependent fragmentation of urinary cfDNA After analyzing the source of urinary cfDNA, we investigated if cfDNA is fragmented as it PT travels through the urinary tract through the analysis of the concentration and size of CE cfDNA DNaseI is highly expressed in the kidney and bladder (http://www.proteinatlas.org/) AC and is present (30) and highly active in urine (31) First we analyzed if the total urinary cfDNA concentration would change if the urine is subjected to in vitro incubation at 37 °C to mimic the time effect on the in vivo passage of urine through the urinary tract and storage in the bladder (see Supplementary Materials and Methods) We collected up to 200 ml urine via percutaneous nephrostomy in patients with ureteric stones and from voided urine from normal controls Urine collected via percutaneous nephrostomy was channeled directly from the kidney pelvis, without passing through the urinary tract The concentration of cfDNA was quantified at various time points ACCEPTED MANUSCRIPT using qPCRr with a 62 bp amplicon in the LEP gene region The cfDNA concentration varied from 52-30,043 GE/ml (10-16,691 GE/ml/mmol Cr) at the time of collection In all cases, the concentration of cfDNA decreased with in vitro incubation at 37 °C under first order kinetics (R2 =0.73-0.99) with a half-life of 2.6-5.1 hours (Fig 2) SC RI PT We then performed sequencing on these samples to i) look for differences in the size of urinary cfDNA fragments in the kidney pelvis and voided urine, and ii) assess if the in vitro incubation process would alter the size of cfDNA fragments We obtained paired renal pelvis and voided urine from a patient who suffered from a 2cm NU ureteric stone causing complete obstruction of the right-sided urinary system We obtained MA paired samples seven days (Fig 3A) and 42 days (Fig 3B) after percutaneous nephrostomy insertion and on both occasions, urine from the renal pelvis had a larger ED proportion of long fragments In vitro incubation of the renal pelvis urine (corresponding to Fig A) showed that the size profiles of these samples displayed a progressive reduction PT in the proportion of long fragments and an increase in amplitude of the 10 bp periodicity in the 50-80 bp region (Fig C and D) The 10 bp periodicity was most pronounced between CE 50 and 80 bp range The amplitude of the periodicity could be represented by the AC difference in the sum of the frequency between the peak lengths at 50, 60 and 70 bp and the sum of the frequency at trough lengths 55, 65 and 75 bp Using the 31 transplant urine samples, the median fragment length is inversely correlated with the periodicity index (Fig 4) which further suggests that a large amplitude of the10bp periodicity is associated with shorter cfDNA fragments The size profiles in renal transplant and HSCT patients resembled that seen in the voided urine in Fig and there was no detectable difference in the size profiles between donor ACCEPTED MANUSCRIPT While hematopoietic cells are consistently the predominant contributor to plasma cfDNA which is present at relatively stable concentrations (22,28), the quantity and composition of urinary cfDNA is highly variable This degree of compositional variation compounds the variation in the concentration of total cfDNA in urine, even after adjustment with concurrent SC RI PT urine creatinine levels This may explain why an assay aimed solely at the sensitive detection of cfDNA from a single source may encounter samples with undetectable levels In our cohort of renal transplant patients, the cfDNA proportional contribution from the kidney and total concentration of kidney-derived cfDNA did not correlate with plasma creatinine levels Serial monitoring of the cfDNA in the urine in renal transplant patients NU from day one to 70 post-transplant shows that longitudinal changes of renal cfDNA MA proportion may reflect transplant allograft health ED This work highlights the difference between the contents of plasma being maintained at a homeostatic equilibrium, while the contents of voided urine is the excretory by-product of PT homeostatic requirements after the one time, unidirectional passage through the urinary system While varying hydration status could conceivably affect total cfDNA concentration, AC tissue CE the dilutional effects cannot account for the variation in proportional contribution from each In vitro incubation experiments suggest that cfDNA in urine collected from the renal pelvis and in spontaneously voided samples are degraded under first order kinetics with a halflife of 2.6-5.1 hours The size profile cfDNA from the renal pelvis shows a larger proportion of long fragments compared with voided urine, although this may be due to the presence of infection or physiological changes in the post-obstructive state in patients with large ureteric stones The reduction of cfDNA concentration during incubation at 37 °C is 11 ACCEPTED MANUSCRIPT reflected in the size profile by a reduced proportion of long fragments and the accentuation of the 10 bp periodicity in the 50-80 bp range The 10 bp periodicity observed in urinary cfDNA is reminiscent of that seen in plasma (32,34), albeit at a larger amplitude in urine compared with plasma The incubation of urine demonstrates that the amplitude of the 10 bp periodicity of cfDNA is due to the time-dependent fragmentation, whereas the SC RI PT mechanism behind the 10 bp periodicity observed in plasma is unclear Although this degradation process affects the global methylation density and also causes fluctuation in the methylation deconvolution results, the high degree of correlation observed in the voided urine of transplant patients suggests that the methylation MA NU deconvolution process is robust despite in vivo degradation In conclusion, this work has provided a bird’s-eye view of the tissue of origin of urinary ED cfDNA The data generated from this work would provide a foundation for further development of genomic and methylomic approaches for urinary cfDNA-based molecular PT diagnostics A similar approach can also be applied to many other bodily fluids of CE importance in clinical medicine, e.g pleural fluid AC Acknowledgements This work is supported by the Hong Kong Research Grants Council under the Themebased research scheme from the Government of the Hong Kong SAR (T12-403/15-N and T12-404/11) We thank Alice Cheng, Lisa Chan, Yongjie Jin, Kam Wing Chan, Patty Tse, Queenie Fung and Mei Shan Cheng for their technical assistance 12 ACCEPTED MANUSCRIPT References Stroun M, Maurice P, Vasioukhin V, Lyautey J, Lederrey C, Lefort F, et al The origin and mechanism of circulating DNA Ann N Y Acad Sci 2000;906:161–8 Lo YM, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CW, et al SC RI PT Presence of fetal DNA in maternal plasma and serum Lancet 1997;350:485–7 Chen XQ, Stroun M, Magnenat JL, Nicod LP, Kurt AM, Lyautey J, et al Microsatellite alterations in plasma DNA of small cell lung cancer patients Nat Med Lo YM, Tein MS, Pang CC, Yeung CK, Tong KL, Hjelm NM Presence of donor- MA NU 1996;2:1033–5 specific DNA in plasma of kidney and liver-transplant recipients Lancet (London, ED England) 1998;351:1329–30 Chiu RWK, Chan KCA, Gao Y, Lau VYM, Zheng W, Leung TY, et al Noninvasive PT prenatal diagnosis of fetal 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plasma and serum after sex-mismatched bone marrow transplantation Clin Chem 2002;48:421–7 29 Hodges E, Molaro A, Dos Santos CO, Thekkat P, Song Q, Uren PJ, et al Directional DNA methylation changes and complex intermediate states accompany lineage specificity in the adult hematopoietic compartment Mol Cell 2011;44:17–28 30 Ito K, Minamiura N, Yamamoto T Human urine DNase I: immunological identity with human pancreatic DNase I, and enzymic and proteochemical properties of the enzyme J Biochem 1984;95:1399–406 16 ACCEPTED MANUSCRIPT 31 Nadano D, Yasuda T, Kishi K Measurement of deoxyribonuclease I activity in human tissues and body fluids by a single radial enzyme-diffusion method Clin Chem 1993;39:448–52 32 Snyder MW, Kircher M, Hill AJ, Daza RM, Shendure J Cell-free DNA Comprises an In Vivo Nucleosome Footprint that Informs Its Tissues-Of-Origin Cell 2016;164:57– 33 SC RI PT 68 Gaffney DJ, McVicker G, Pai AA, Fondufe-Mittendorf YN, Lewellen N, Michelini K, et al Controls of nucleosome positioning in the human genome PLoS Genet Public Library of Science; 2012;8:e1003036 NU Lo YMD, Chan KCA, Sun H, Chen EZ, Jiang P, Lun FMF, et al Maternal plasma MA DNA sequencing reveals the genome-wide genetic and mutational profile of the CE PT ED fetus Sci Transl Med 2010;2:61ra91 AC 34 17 ACCEPTED MANUSCRIPT Tables Table General information and methylation deconvolution results for urinary cfDNA 31 samples from renal transplant and HSCT patients Each sample ID represents urine collected from a patient at single time point A and B denote two samples collected at T P I R different times on the same day from the same patient The urine creatinine from each urine sample and the concurrent plasma creatinine results are displayed Transplant type Kidney (%) Urothelium (%) Neutrophils (%) T10 Renal 97.2 0.0 0.0 T11 Renal 0.7 8.5 85.4 T18 Renal 6.5 9.0 T20 Renal 1.4 8.4 T29 Renal 100.0 0.0 T34 Renal 43.2 16.2 T35 Renal 14.1 T40 Renal 100.0 T42 Renal 36.9 T50 Renal 31.8 T51 Renal T53 Renal T54 AC 3.8 T-cells (%) B-cells (%) Donor SNP (%) GE/ml urine Urine creatinine (mmol/L) Plasma creatinine (mmol/L) CpG Methylation density (%) 2.8 81.1 974 18.2 262 68.6 2.5 3.0 2.9 7,038 3.0 178 69.2 M 79.2 D E 4.0 1.3 9.3 2,531 3.6 186 71.8 83.2 5.2 1.8 2.5 25,710 5.7 203 70.4 0.0 0.0 0.0 89.1 1,489 13.3 142 69.5 39.0 0.0 1.6 39.4 1,113 3.0 188 70.7 PT E C N A C S U 0.0 78.7 0.0 3.4 4.1 13,088 8.3 107 69.6 0.0 0.0 0.0 0.0 94.0 1,372 6.0 129 64.8 11.4 50.5 0.0 1.2 34.3 559 1.7 92 64.5 64.0 0.0 0.0 4.2 32.9 1,412 7.9 957 73.0 46.8 35.8 12.6 3.7 1.1 53.2 1,864 4.7 932 73.5 16.5 13.8 65.4 0.0 4.3 16.7 1,265 12.0 478 67.1 Renal 8.5 8.3 79.1 1.7 2.3 10.9 15,033 4.4 337 69.1 T56 Renal 13.0 20.8 59.3 6.9 0.0 8.9 18,211 4.0 327 69.4 T57 Renal 1.3 13.8 80.0 1.7 3.1 4.8 1,342 8.2 368 65.3 18 ACCEPTED MANUSCRIPT T63 Renal 5.7 5.5 83.7 2.1 3.0 4.4 20,074 3.3 250 70.5 T64 Renal 1.4 6.0 85.6 4.5 2.5 1.5 9,162 6.1 350 68.9 T65 Renal 6.0 5.0 82.0 4.9 2.1 5.0 21,372 3.4 249 71.3 T69 Renal 99.3 0.0 0.0 0.0 0.7 88.0 3,585 4.5 281 66.3 T72 Renal 5.7 12.0 72.0 7.8 2.6 4.6 3,567 5.8 194 70.8 T75_A Renal 99.1 0.0 0.0 0.0 0.9 78.8 1,046 8.3 288 68.0 T75_B Renal 99.6 0.0 0.0 0.0 0.4 77.0 1,401 10.8 288 67.8 T80_A Renal 100.0 0.0 0.0 0.0 0.0 87.4 2,919 13.5 315 68.2 T80_B Renal 100.0 0.0 0.0 0.0 0.0 85.2 4,659 4.3 315 68.7 T88 Renal 30.0 16.3 49.0 2.9 1.7 16.1 3,135 4.7 136 66.9 T89 Renal 6.6 4.1 87.0 0.3 2.0 2,522 2.3 216 72.3 T45 HSCT 29.6 0.0 60.5 8.5 1.4 77.6 376 6.4 78 64.1 T85 HSCT 100.0 0.0 0.0 0.0 0.0 6.0 225 4.3 78 61.1 T86 HSCT 100.0 0.0 0.0 0.0 0.0 11.4 559 8.1 75 66.4 T102_A HSCT 100.0 0.0 0.0 0.0 0.0 6.2 165 6.9 75 67.2 T102_B HSCT 100.0 0.0 0.0 0.0 0.0 6.9 510 11 75 69.4 D E U N A M T P E C C A 19 I R SC 4.1 T P ACCEPTED MANUSCRIPT Figure Legends Fig The proportional contributions of cfDNA derived from the blood cells and kidney in 31 urine samples from HSCT (triangles) and renal transplant (round dots) patients The x-axis displays the percentage contribution from the donor tissue as SC RI PT calculated from donor-specific SNPs, and the y-axis displays the percentage contribution of the donor tissue as deduced by methylation deconvolution There is a high correlation between the percentage contribution as determined by methylation deconvolution and NU SNP analysis, with a R2 of 0.97 Fig The concentration of urinary cfDNA during the 37°C in vitro incubation MA experiments for urine from the renal pelvis (A-C) and voided urine (D-F) The x-axis is the concentration of cfDNA in GE/mL urine displayed in a log scale and the y-axis is the ED incubation time in hours A-C represent the incubation of urine from three patients with ureteric stones with percutaneous nephrostomy inserted D-F repesent the incubation of PT urine voided by three normal control subjects cfDNA is degraded under first order kinetics CE in both renal pelvis and void urine (R2=0.73-0.99) with a half-life of 2.6 – 5.1 hours AC Fig Size profiles of urinary DNA from voided urine, the renal pelvis, and during in vitro incubation at 37°C (A) and (B) show the size profiles of urinary cfDNA from paired samples from the renal pelvis and voided urine on two occasions 35 days apart The x-axis displays the size in single base pair resolution and the y-axis shows the frequency at each length Red lines represent voided urine produced by the left kidney The blue lines represent urine from the right renal pelvis collected simultaneously Urine from the renal pelvis has a larger proportion of long cfDNA fragments (C) and (D) are the size profiles of cfDNA from urine from the renal pelvis at 0, and hours of in vitro incubation at 37°C 20 ACCEPTED MANUSCRIPT (C) displays the frequency at each base pair length, and (D) displays the cumulative frequency Blue, green and red lines represent the size profiles of cfDNA at the time of collection, after hours and after hours of incubation, respectively The periodicity index of the urine from the renal pelvis increased from 2.0 to 3.4 and 4.7 at 0, and hours respectively These graphs illustrate the increase in the amplitude of the 10 bp periodicity SC RI PT and a reduction in the proportion of long cfDNA fragments with in vitro incubation Fig Correlation between the periodicity index and median cfDNA length in 31 voided urine samples The median cfDNA fragment length is plotted against the periodicity index for the voided urine samples from renal and HSCT patients There is a AC CE PT ED MA NU negative correlation between the median fragment length and the periodicity index 21 AC CE PT ED MA NU SC RI PT ACCEPTED MANUSCRIPT 22 AC CE PT ED MA NU SC RI PT ACCEPTED MANUSCRIPT 23 AC CE PT ED MA NU SC RI PT ACCEPTED MANUSCRIPT 24 AC Highlights CE PT ED MA NU SC RI PT ACCEPTED MANUSCRIPT Genomewide methylation signatures can infer the tissue of origin of urinary cfDNA Large variation in the proportional contribution of cfDNA from different tissues Urinary cfDNA is fragmented under first-order kinetics in a time dependent manner 25