Urinary cell-free DNA is a versatile analyte for monitoring infections of the urinary tract

Burnham, Philip; Dadhania, Darshana; Heyang, Michael; Chen, Fanny; Westblade, Lars F.; Suthanthiran, Manikkam; Lee, John Richard; Vlaminck, Iwijn De

doi:10.1038/s41467-018-04745-0

Cited by 144 publications

(178 citation statements)

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“…samples. We and others have identified ddcfDNA as a non-invasive, quantitative marker of graft 16 injury in solid-organ transplantation 2,4,30,31 . For sex-mismatched donor-recipient pairs, the 17…”

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confidence: 96%

“…Second, 23 the assay quantifies the relative abundance of microbes via WGBS of cfDNA. Several studies 24 have investigated the utility of conventional, metagenomic sequencing of cfDNA for infection 25 testing in clinical samples 2,4,10,11 . We show here that WGBS is compatible with such analyses.…”

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confidence: 99%

“…proportion of ddcfDNA can be estimated by evaluating the coverage of the Y chromosome relative 18 to the autosomal chromosomes (see Methods) 2,32 . We verified that the proportion of ddcfDNA 19 measured by sequencing of bisulfite-treated cfDNA matched the proportion of ddcfDNA measured 20 using conventional sequencing (n=36 matched samples, Spearman's rho = 0.97, p-value < 21 2.2x10 -16 , see Methods, supplemental figure 2), and then quantified the proportion of ddcfDNA 22 in urine for all sex-mismatched donor-recipient transplant pairs (n=46).…”

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confidence: 99%

“…to conventional metagenomic cfDNA assays 2,41 , this assay requires a single additional 12 experimental step, bisulfite treatment of the cfDNA isolate, which is inexpensive and can be 13…”

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confidence: 99%

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A cell-free DNA metagenomic sequencing assay that integrates the damage response to infection

Cheng

Burnham

Lee

et al. 2019

Preprint

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High-throughput metagenomic sequencing offers an unbiased approach to identify pathogens in clinical samples. Conventional metagenomic sequencing however does not integrate information about the host, which is often critical to distinguish infection from infectious disease, and to assess the severity of disease. Here, we explore the utility of high-throughput sequencing of cell-free DNA after bisulfite conversion to map the tissue and cell types of origin of host-derived cell-free DNA, and to profile the bacterial and viral metagenome. We applied this assay to 51 urinary cfDNA isolates collected from a cohort of kidney transplant recipients with and without bacterial and viral infection of the urinary tract. We find that the cell and tissue types of origin of urinary cell-free DNA can be derived from its genome-wide profile of methylation marks, and strongly depend on infection status. We find evidence of kidney and bladder tissue damage due to viral and bacterial infection, respectively, and of the recruitment of neutrophils to the urinary tract during infection. Through direct comparison to conventional metagenomic sequencing as well as clinical tests of infection, we find this assay accurately captures the bacterial and viral composition of the sample. The assay presented here is straightforward to implement, offers a systems view into bacterial and viral infections of the urinary tract, and can find future use as a tool for the differential diagnosis of infections.Affiliations:

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confidence: 96%

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confidence: 99%

mentioning

confidence: 99%

“…to conventional metagenomic cfDNA assays 2,41 , this assay requires a single additional 12 experimental step, bisulfite treatment of the cfDNA isolate, which is inexpensive and can be 13…”

mentioning

confidence: 99%

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A cell-free DNA metagenomic sequencing assay that integrates the damage response to infection

Cheng

Burnham

Lee

et al. 2019

Preprint

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“…Metagenomic sequencing of cfDNA offers a highly sensitive approach to screen for pathogens in clinical 3 samples [1][2][3][4] . The sensitivity of metagenomic sequencing of cfDNA in plasma can be boosted by the 4 implementation of library preparations optimized to recover short, degraded microbial cfDNA 5 , or by 5 strategies that selectively enrich microbial DNA or deplete host DNA [6][7][8] .…”

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confidence: 99%

Separating the signal from the noise in metagenomic cell-free DNA sequencing

Burnham

Gomez‐Lopez

Heyang

et al. 2019

Preprint

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Cell-free DNA (cfDNA) in blood, urine and other biofluids provides a unique window into 24 human health. A proportion of cfDNA is derived from bacteria and viruses, creating opportunities for the 25 diagnosis of infection via metagenomic sequencing. The total biomass of microbial-derived cfDNA in 26 clinical isolates is low, which makes metagenomic cfDNA sequencing susceptible to contamination and 27 alignment noise. Here, we report Low Biomass Background Correction (LBBC), a bioinformatics noise 28 filtering tool informed by the uniformity of the coverage of microbial genomes and the batch variation in 29 the absolute abundance of microbial cfDNA. We demonstrate that LBBC leads to a dramatic reduction in 30 false positive rate while minimally affecting the true positive rate for a cfDNA test to screen for urinary 31 tract infection. We next performed high throughput sequencing of cfDNA in amniotic fluid collected from 32 term uncomplicated pregnancies or those complicated with clinical chorioamnionitis with and without intra-33 amniotic infection. The data provide unique insight into the properties of fetal and maternal cfDNA in 34 amniotic fluid, demonstrate the utility of cfDNA to screen for intra-amniotic infection, support the view 35 that the amniotic fluid is sterile during normal pregnancy, and reveal cases of intra-amniotic inflammation 36 without infection at term. 37 38 2 Introduction 1 2Metagenomic sequencing of cfDNA offers a highly sensitive approach to screen for pathogens in clinical 3 samples 1-4 . The sensitivity of metagenomic sequencing of cfDNA in plasma can be boosted by the 4 implementation of library preparations optimized to recover short, degraded microbial cfDNA 5 , or by 5 strategies that selectively enrich microbial DNA or deplete host DNA 6-8 . A major remaining challenge is 6the relatively poor specificity of the cfDNA metagenomic sequencing, which is limited by alignment noise, 7annotation errors in reference genomes and environmental contamination 9 . 8 9Here, we report low biomass background correction (LBBC), a tool to filter background contamination and 10 noise in cfDNA metagenomic sequencing datasets. We have applied LBBC to two independent datasets. 11We first re-analyzed a dataset from a previous study that investigated the utility of urinary cfDNA as an 12analyte to monitor urinary tract infection 2 (UTI). Next, we generated a new dataset of cfDNA in amniotic 13 fluid collected from uncomplicated pregnancies or those complicated with clinical chorioamnionitis at term, 14a common heterogeneous condition that can occur in the presence or absence of intra-amniotic infection 10 . 15We report a first, detailed study of the properties of cfDNA in amniotic fluid. For both datasets, detailed 16 microbiologic workups, including results from conventional bacterial culture and/or 16S rRNA sequencing, 17were available to benchmark the LBBC workflow. We demonstrate that LBBC greatly improves the 18 specificity of cfDNA metagenomic sequencing, while minimally affecting its sensitivity. ...

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Selective Enrichment of Hypermethylated DNA by a Multivalent Binding Platform

Kolkman

Segerink

Huskens

2022

Adv Materials Inter

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One of the key aspects of surviving cancer is to detect the disease as early as possible, thereby enlarging the window of opportunity of curing it. [4,5] A recent trend for early cancer diagnostics is the pre-symptomatic detection of cancer biomarkers in liquid biopsy samples and, therefore, improving the survival rate. [6,7] Hypermethylated DNA (hmDNA) is one of the typical biomarkers found in liquid biopsy samples of cancer patients. [8,9] The presence of hmDNA in blood and urine is correlated to multiple types of cancer, including gastric, lung and ovarian cancer. [10][11][12][13] From a genetic point of view, DNA methylation takes place predominantly at promotor regions with a relative high amount of cytosine bases that are followed directly by a guanine (CpGs) in the 5′-to-3′ direction, the so-called CpG-rich regions. [13] The methylation of a CpG is an epigenetic alternation in which a methyl group is covalently bonded to the cytosine base at the fifth carbon. By CpG methylation, gene expressions are controlled in cells. As a consequence, methylation can play a role in tumor development when tumor suppressor genes are methylated, thereby repressing the transcription and thus silencing these genes. [14] Hypermethylation refers to the situation that methylation of a promotor region occurs, which in a healthy situation does not occur.Early disease detection as well as disease progress and the effectiveness of a therapy can be monitored by measuring the hmDNA concentration over time. [15] Yet, the concentration of hmDNA, especially in early-stage cancers, can be as low as a few DNA copies per liquid biopsy sample. [16][17][18] The current approach to measure hmDNA employs DNA isolation and bisulfite conversion, making it time-consuming and labor intensive. As a result, the method is not widely applicable in the clinic. Typically, hmDNA is detected in bisulfite-treated DNA samples by quantitative polymerase chain reaction (PCR) (qPCR, of the region of interest; can be cancer dependent). Alternative hmDNA detection approaches that may be suitable for use in point-of-care applications, include electrochemistry, [19] CRISPR [20] and isothermal amplification. [21] The detection of specific hmDNA biomarkers by sequencing or biosensing approaches involves the ability to distinguish between hmDNA and non-methylated DNA. [22][23][24] Commonly a preselection step is applied, and three different approaches exist in order to differentiate between hmDNA and non-methylatedThe preselection of hypermethylated DNA (hmDNA) from liquid biopsy samples is key to enable early-stage cancer diagnostics. Due to limited selectivity of the existing preselection approaches, however, wide integration in the clinic is currently prohibited. Here, it is argued that an affinity method on a surface, such as used in affinity chromatography, can be significantly improved by employing the principles of multivalency and superselectivity. In the here proposed method, a methyl binding domain (MBD) protein immobilized at a surface is used as a recepto...

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Urinary cell-free DNA is a versatile analyte for monitoring infections of the urinary tract

Cited by 144 publications

References 59 publications

A cell-free DNA metagenomic sequencing assay that integrates the damage response to infection

A cell-free DNA metagenomic sequencing assay that integrates the damage response to infection

Separating the signal from the noise in metagenomic cell-free DNA sequencing

Selective Enrichment of Hypermethylated DNA by a Multivalent Binding Platform

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