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Characterizing the molecular composition and diagnostic potential of Mycobacterium tuberculosis urinary cell-free DNA using next-generation sequencing

BACKGROUND: Urine cell-free DNA (cfDNA) is an attractive target for diagnosing pulmonary Mycobacterium tuberculosis (MTB) infection, but has not been thoroughly characterized as a biomarker. METHODS: This study was performed to investigate the size and composition of urine cfDNA from tuberculosis (T...

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Autores principales: Oreskovic, Amy, Waalkes, Adam, Holmes, Elizabeth A., Rosenthal, Christopher A., Wilson, Douglas P.K., Shapiro, Adrienne E., Drain, Paul K., Lutz, Barry R., Salipante, Stephen J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8627387/
https://www.ncbi.nlm.nih.gov/pubmed/34562627
http://dx.doi.org/10.1016/j.ijid.2021.09.042
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author Oreskovic, Amy
Waalkes, Adam
Holmes, Elizabeth A.
Rosenthal, Christopher A.
Wilson, Douglas P.K.
Shapiro, Adrienne E.
Drain, Paul K.
Lutz, Barry R.
Salipante, Stephen J.
author_facet Oreskovic, Amy
Waalkes, Adam
Holmes, Elizabeth A.
Rosenthal, Christopher A.
Wilson, Douglas P.K.
Shapiro, Adrienne E.
Drain, Paul K.
Lutz, Barry R.
Salipante, Stephen J.
author_sort Oreskovic, Amy
collection PubMed
description BACKGROUND: Urine cell-free DNA (cfDNA) is an attractive target for diagnosing pulmonary Mycobacterium tuberculosis (MTB) infection, but has not been thoroughly characterized as a biomarker. METHODS: This study was performed to investigate the size and composition of urine cfDNA from tuberculosis (TB) patients with minimal bias using next-generation sequencing (NGS). A combination of DNA extraction and single-stranded sequence library preparation methods demonstrated to recover short, highly degraded cfDNA fragments was employed. Urine cfDNA from 10 HIV-positive patients with pulmonary TB and two MTB-negative controls was examined. RESULTS: MTB-derived cfDNA was identifiable by NGS from all MTB-positive patients and was absent from negative controls. MTB cfDNA was significantly shorter than human cfDNA, with median fragment lengths of ≤19–52 bp and 42–92 bp, respectively. MTB cfDNA abundance increased exponentially with decreased fragment length, having a peak fragment length of ≤19 bp in most samples. In addition, we identified a larger fraction of short human genomic cfDNA, ranging from 29 to 53 bp, than previously reported. Urine cfDNA fragments spanned the MTB genome with relative uniformity, but nucleic acids derived from multicopy elements were proportionately over-represented. CONCLUSIONS: TB urine cfDNA is a potentially powerful biomarker but is highly fragmented, necessitating special procedures to maximize its recovery and detection.
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spelling pubmed-86273872021-12-03 Characterizing the molecular composition and diagnostic potential of Mycobacterium tuberculosis urinary cell-free DNA using next-generation sequencing Oreskovic, Amy Waalkes, Adam Holmes, Elizabeth A. Rosenthal, Christopher A. Wilson, Douglas P.K. Shapiro, Adrienne E. Drain, Paul K. Lutz, Barry R. Salipante, Stephen J. Int J Infect Dis Article BACKGROUND: Urine cell-free DNA (cfDNA) is an attractive target for diagnosing pulmonary Mycobacterium tuberculosis (MTB) infection, but has not been thoroughly characterized as a biomarker. METHODS: This study was performed to investigate the size and composition of urine cfDNA from tuberculosis (TB) patients with minimal bias using next-generation sequencing (NGS). A combination of DNA extraction and single-stranded sequence library preparation methods demonstrated to recover short, highly degraded cfDNA fragments was employed. Urine cfDNA from 10 HIV-positive patients with pulmonary TB and two MTB-negative controls was examined. RESULTS: MTB-derived cfDNA was identifiable by NGS from all MTB-positive patients and was absent from negative controls. MTB cfDNA was significantly shorter than human cfDNA, with median fragment lengths of ≤19–52 bp and 42–92 bp, respectively. MTB cfDNA abundance increased exponentially with decreased fragment length, having a peak fragment length of ≤19 bp in most samples. In addition, we identified a larger fraction of short human genomic cfDNA, ranging from 29 to 53 bp, than previously reported. Urine cfDNA fragments spanned the MTB genome with relative uniformity, but nucleic acids derived from multicopy elements were proportionately over-represented. CONCLUSIONS: TB urine cfDNA is a potentially powerful biomarker but is highly fragmented, necessitating special procedures to maximize its recovery and detection. Elsevier 2021-11 /pmc/articles/PMC8627387/ /pubmed/34562627 http://dx.doi.org/10.1016/j.ijid.2021.09.042 Text en © 2021 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Oreskovic, Amy
Waalkes, Adam
Holmes, Elizabeth A.
Rosenthal, Christopher A.
Wilson, Douglas P.K.
Shapiro, Adrienne E.
Drain, Paul K.
Lutz, Barry R.
Salipante, Stephen J.
Characterizing the molecular composition and diagnostic potential of Mycobacterium tuberculosis urinary cell-free DNA using next-generation sequencing
title Characterizing the molecular composition and diagnostic potential of Mycobacterium tuberculosis urinary cell-free DNA using next-generation sequencing
title_full Characterizing the molecular composition and diagnostic potential of Mycobacterium tuberculosis urinary cell-free DNA using next-generation sequencing
title_fullStr Characterizing the molecular composition and diagnostic potential of Mycobacterium tuberculosis urinary cell-free DNA using next-generation sequencing
title_full_unstemmed Characterizing the molecular composition and diagnostic potential of Mycobacterium tuberculosis urinary cell-free DNA using next-generation sequencing
title_short Characterizing the molecular composition and diagnostic potential of Mycobacterium tuberculosis urinary cell-free DNA using next-generation sequencing
title_sort characterizing the molecular composition and diagnostic potential of mycobacterium tuberculosis urinary cell-free dna using next-generation sequencing
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8627387/
https://www.ncbi.nlm.nih.gov/pubmed/34562627
http://dx.doi.org/10.1016/j.ijid.2021.09.042
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