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1.
Proc Natl Acad Sci U S A ; 119(4)2022 01 25.
Article En | MEDLINE | ID: mdl-35058359

Allogeneic hematopoietic cell transplantation (HCT) provides effective treatment for hematologic malignancies and immune disorders. Monitoring of posttransplant complications is critical, yet current diagnostic options are limited. Here, we show that cell-free DNA (cfDNA) in blood is a versatile analyte for monitoring of the most important complications that occur after HCT: graft-versus-host disease (GVHD), a frequent immune complication of HCT, infection, relapse of underlying disease, and graft failure. We demonstrate that these therapeutic complications are informed from a single assay, low-coverage bisulfite sequencing of cfDNA, followed by disease-specific bioinformatic analyses. To inform GVHD, we profile cfDNA methylation marks to trace the cfDNA tissues-of-origin and to quantify tissue-specific injury. To inform infection, we implement metagenomic cfDNA profiling. To inform cancer relapse, we implement analyses of tumor-specific genomic aberrations. Finally, to detect graft failure, we quantify the proportion of donor- and recipient-specific cfDNA. We applied this assay to 170 plasma samples collected from 27 HCT recipients at predetermined timepoints before and after allogeneic HCT. We found that the abundance of solid-organ-derived cfDNA in the blood at 1 mo after HCT is predictive of acute GVHD (area under the curve, 0.88). Metagenomic profiling of cfDNA revealed the frequent occurrence of viral reactivation in this patient population. The fraction of donor-specific cfDNA was indicative of relapse and remission, and the fraction of tumor-specific cfDNA was informative of cancer relapse. This proof-of-principle study shows that cfDNA has the potential to improve the care of allogeneic HCT recipients by enabling earlier detection and better prediction of the complex array of complications that occur after HCT.


Cell-Free Nucleic Acids , DNA Fingerprinting , Graft vs Host Disease/diagnosis , Graft vs Host Disease/etiology , Hematopoietic Stem Cell Transplantation/adverse effects , Biomarkers , DNA Methylation , Disease Progression , Graft vs Host Disease/blood , Hematopoietic Stem Cell Transplantation/methods , Humans , Liquid Biopsy/methods , Organ Specificity/genetics , Postoperative Complications/blood , Postoperative Complications/diagnosis , Postoperative Complications/etiology , Recurrence , Transplantation, Homologous
2.
bioRxiv ; 2021 Nov 23.
Article En | MEDLINE | ID: mdl-34845444

Metagenomic DNA sequencing is a powerful tool to characterize microbial communities but is sensitive to environmental DNA contamination, in particular when applied to samples with low microbial biomass. Here, we present contamination-free metagenomic DNA sequencing (Coffee-seq), a metagenomic sequencing assay that is robust against environmental contamination. The core idea of Coffee-seq is to tag the DNA in the sample prior to DNA isolation and library preparation with a label that can be recorded by DNA sequencing. Any contaminating DNA that is introduced in the sample after tagging can then be bioinformatically identified and removed. We applied Coffee-seq to screen for infections from microorganisms with low burden in blood and urine, to identify COVID-19 co-infection, to characterize the urinary microbiome, and to identify microbial DNA signatures of inflammatory bowel disease in blood.

3.
Med (N Y) ; 2(4): 411-422.e5, 2021 04 09.
Article En | MEDLINE | ID: mdl-33521749

BACKGROUND: Coronavirus disease 2019 (COVID-19) primarily affects the lungs, but evidence of systemic disease with multi-organ involvement is emerging. Here, we developed a blood test to broadly quantify cell-, tissue-, and organ-specific injury due to COVID-19. METHODS: Our test leverages genome-wide methylation profiling of circulating cell-free DNA in plasma. We assessed the utility of this test to identify subjects with severe disease in two independent, longitudinal cohorts of hospitalized patients. Cell-free DNA profiling was performed on 104 plasma samples from 33 COVID-19 patients and compared to samples from patients with other viral infections and healthy controls. FINDINGS: We found evidence of injury to the lung and liver and involvement of red blood cell progenitors associated with severe COVID-19. The concentration of cell-free DNA correlated with the World Health Organization (WHO) ordinal scale for disease progression and was significantly increased in patients requiring intubation. CONCLUSIONS: This study points to the utility of cell-free DNA as an analyte to monitor and study COVID-19. FUNDING: This work was supported by NIH grants 1DP2AI138242 (to I.D.V.), R01AI146165 (to I.D.V., M.P.C., F.M.M., and J.R.), 1R01AI151059 (to I.D.V.), K08-CA230156 (to W.G.), and R33-AI129455 to C.Y.C., a Synergy award from the Rainin Foundation (to I.D.V.), a SARS-CoV-2 seed grant at Cornell (to I.D.V.), a National Sciences and Engineering Research Council of Canada fellowship PGS-D3 (to A.P.C.), and a Burroughs-Wellcome CAMS Award (to W.G.). D.C.V. is supported by a Fonds de la Recherche en Sante du Quebec Clinical Research Scholar Junior 2 award. C.Y.C. is supported by the California Initiative to Advance Precision Medicine, and the Charles and Helen Schwab Foundation.


COVID-19 , Cell-Free Nucleic Acids , Virus Diseases , Humans , Methylation , SARS-CoV-2/genetics
4.
medRxiv ; 2020 Jul 29.
Article En | MEDLINE | ID: mdl-32766608

COVID-19 primarily affects the lungs, but evidence of systemic disease with multi-organ involvement is emerging. Here, we developed a blood test to broadly quantify cell, tissue, and organ specific injury due to COVID-19, using genome-wide methylation profiling of circulating cell-free DNA in plasma. We assessed the utility of this test to identify subjects with severe disease in two independent, longitudinal cohorts of hospitalized patients. Cell-free DNA profiling was performed on 104 plasma samples from 33 COVID-19 patients and compared to samples from patients with other viral infections and healthy controls. We found evidence of injury to the lung and liver and involvement of red blood cell progenitors associated with severe COVID-19. The concentration of cfDNA correlated with the WHO ordinal scale for disease progression and was significantly increased in patients requiring intubation. This study points to the utility of cell-free DNA as an analyte to monitor and study COVID-19.

5.
Microbiome ; 8(1): 18, 2020 02 11.
Article En | MEDLINE | ID: mdl-32046792

BACKGROUND: Cell-free DNA (cfDNA) in blood, urine, and other biofluids provides a unique window into human health. A proportion of cfDNA is derived from bacteria and viruses, creating opportunities for the diagnosis of infection via metagenomic sequencing. The total biomass of microbial-derived cfDNA in clinical isolates is low, which makes metagenomic cfDNA sequencing susceptible to contamination and alignment noise. RESULTS: Here, we report low biomass background correction (LBBC), a bioinformatics noise filtering tool informed by the uniformity of the coverage of microbial genomes and the batch variation in the absolute abundance of microbial cfDNA. We demonstrate that LBBC leads to a dramatic reduction in false positive rate while minimally affecting the true positive rate for a cfDNA test to screen for urinary tract infection. We next performed high-throughput sequencing of cfDNA in amniotic fluid collected from term uncomplicated pregnancies or those complicated with clinical chorioamnionitis with and without intra-amniotic infection. CONCLUSIONS: The data provide unique insight into the properties of fetal and maternal cfDNA in amniotic fluid, demonstrate the utility of cfDNA to screen for intra-amniotic infection, support the view that the amniotic fluid is sterile during normal pregnancy, and reveal cases of intra-amniotic inflammation without infection at term. Video abstract.


Cell-Free Nucleic Acids/analysis , Computational Biology/methods , DNA, Bacterial/analysis , Metagenome , Sequence Analysis, DNA/methods , Amniotic Fluid/microbiology , Cell-Free Nucleic Acids/blood , Cell-Free Nucleic Acids/urine , Chorioamnionitis/microbiology , Communicable Diseases/diagnosis , Communicable Diseases/microbiology , Communicable Diseases/urine , Cross-Sectional Studies , Data Analysis , False Positive Reactions , Female , Fetus/microbiology , High-Throughput Nucleotide Sequencing , Humans , Inflammation , Male , Pregnancy , Pregnancy Complications/diagnosis , Pregnancy Complications/microbiology , Software
6.
Proc Natl Acad Sci U S A ; 116(37): 18738-18744, 2019 09 10.
Article En | MEDLINE | ID: mdl-31451660

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 (cfDNA) after bisulfite conversion to map the tissue and cell types of origin of host-derived cfDNA, 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 cfDNA 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 infection.


Cell-Free Nucleic Acids/isolation & purification , Host-Pathogen Interactions/genetics , Metagenome/genetics , Metagenomics/methods , Postoperative Complications/diagnosis , Urinary Tract Infections/diagnosis , Bacterial Infections/diagnosis , Bacterial Infections/microbiology , Bacterial Infections/urine , Biomarkers/urine , Cell-Free Nucleic Acids/genetics , Cell-Free Nucleic Acids/urine , DNA Methylation/genetics , DNA, Bacterial/genetics , DNA, Bacterial/isolation & purification , DNA, Bacterial/urine , DNA, Viral/genetics , DNA, Viral/isolation & purification , DNA, Viral/urine , Diagnosis, Differential , Female , High-Throughput Nucleotide Sequencing , Host-Pathogen Interactions/immunology , Humans , Kidney/cytology , Kidney/immunology , Kidney/microbiology , Kidney/pathology , Kidney Failure, Chronic/surgery , Kidney Transplantation/adverse effects , Male , Neutrophil Infiltration/immunology , Postoperative Complications/immunology , Postoperative Complications/microbiology , Postoperative Complications/urine , Transplant Recipients , Urinary Bladder/cytology , Urinary Bladder/immunology , Urinary Bladder/microbiology , Urinary Bladder/pathology , Urinary Tract Infections/immunology , Urinary Tract Infections/microbiology , Urinary Tract Infections/urine , Virus Diseases/diagnosis , Virus Diseases/immunology , Virus Diseases/urine , Virus Diseases/virology
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