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Whole genome sequencing of low input circulating cell‐free DNA obtained from normal human subjects

Cell‐free DNA circulates in plasma at low levels as a normal by‐product of cellular apoptosis. Multiple clinical pathologies, as well as environmental stressors can lead to increased circulating cell‐free DNA (ccfDNA) levels. Plasma DNA studies frequently employ targeted amplicon deep sequencing pla...

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Autores principales: Foley, Julie F., Elgart, Brian, Alex Merrick, B., Phadke, Dhiral P., Cook, Molly E., Malphurs, Jason A., Solomon, Gregory G., Shah, Ruchir R., Fessler, Michael B., Miller, Frederick W., Gerrish, Kevin E.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8339531/
https://www.ncbi.nlm.nih.gov/pubmed/34350716
http://dx.doi.org/10.14814/phy2.14993
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author Foley, Julie F.
Elgart, Brian
Alex Merrick, B.
Phadke, Dhiral P.
Cook, Molly E.
Malphurs, Jason A.
Solomon, Gregory G.
Shah, Ruchir R.
Fessler, Michael B.
Miller, Frederick W.
Gerrish, Kevin E.
author_facet Foley, Julie F.
Elgart, Brian
Alex Merrick, B.
Phadke, Dhiral P.
Cook, Molly E.
Malphurs, Jason A.
Solomon, Gregory G.
Shah, Ruchir R.
Fessler, Michael B.
Miller, Frederick W.
Gerrish, Kevin E.
author_sort Foley, Julie F.
collection PubMed
description Cell‐free DNA circulates in plasma at low levels as a normal by‐product of cellular apoptosis. Multiple clinical pathologies, as well as environmental stressors can lead to increased circulating cell‐free DNA (ccfDNA) levels. Plasma DNA studies frequently employ targeted amplicon deep sequencing platforms due to limited concentrations (ng/ml) of ccfDNA in the blood. Here, we report whole genome sequencing (WGS) and read distribution across chromosomes of ccfDNA extracted from two human plasma samples from normal, healthy subjects, representative of limited clinical samples at <1 ml. Amplification was sufficiently robust with ~90% of the reference genome (GRCh38.p2) exhibiting 10X coverage. Chromosome read coverage was uniform and directly proportional to the number of reads for each chromosome across both samples. Almost 99% of the identified genomic sequence variants were known annotated dbSNP variants in the hg38 reference genome. A high prevalence of C>T and T>C mutations was present along with a strong concordance of variants shared between the germline genome databases; gnomAD (81.1%) and the 1000 Genome Project (93.6%). This study demonstrates isolation and amplification procedures from low input ccfDNA samples that can detect sequence variants across the whole genome from amplified human plasma ccfDNA that can translate to multiple clinical research disciplines.
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spelling pubmed-83395312021-08-11 Whole genome sequencing of low input circulating cell‐free DNA obtained from normal human subjects Foley, Julie F. Elgart, Brian Alex Merrick, B. Phadke, Dhiral P. Cook, Molly E. Malphurs, Jason A. Solomon, Gregory G. Shah, Ruchir R. Fessler, Michael B. Miller, Frederick W. Gerrish, Kevin E. Physiol Rep Original Articles Cell‐free DNA circulates in plasma at low levels as a normal by‐product of cellular apoptosis. Multiple clinical pathologies, as well as environmental stressors can lead to increased circulating cell‐free DNA (ccfDNA) levels. Plasma DNA studies frequently employ targeted amplicon deep sequencing platforms due to limited concentrations (ng/ml) of ccfDNA in the blood. Here, we report whole genome sequencing (WGS) and read distribution across chromosomes of ccfDNA extracted from two human plasma samples from normal, healthy subjects, representative of limited clinical samples at <1 ml. Amplification was sufficiently robust with ~90% of the reference genome (GRCh38.p2) exhibiting 10X coverage. Chromosome read coverage was uniform and directly proportional to the number of reads for each chromosome across both samples. Almost 99% of the identified genomic sequence variants were known annotated dbSNP variants in the hg38 reference genome. A high prevalence of C>T and T>C mutations was present along with a strong concordance of variants shared between the germline genome databases; gnomAD (81.1%) and the 1000 Genome Project (93.6%). This study demonstrates isolation and amplification procedures from low input ccfDNA samples that can detect sequence variants across the whole genome from amplified human plasma ccfDNA that can translate to multiple clinical research disciplines. John Wiley and Sons Inc. 2021-08-04 /pmc/articles/PMC8339531/ /pubmed/34350716 http://dx.doi.org/10.14814/phy2.14993 Text en © 2021 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society. This article has been contributed to by US Government employees and their work is in the public domain in the USA. https://creativecommons.org/licenses/by/4.0/This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
spellingShingle Original Articles
Foley, Julie F.
Elgart, Brian
Alex Merrick, B.
Phadke, Dhiral P.
Cook, Molly E.
Malphurs, Jason A.
Solomon, Gregory G.
Shah, Ruchir R.
Fessler, Michael B.
Miller, Frederick W.
Gerrish, Kevin E.
Whole genome sequencing of low input circulating cell‐free DNA obtained from normal human subjects
title Whole genome sequencing of low input circulating cell‐free DNA obtained from normal human subjects
title_full Whole genome sequencing of low input circulating cell‐free DNA obtained from normal human subjects
title_fullStr Whole genome sequencing of low input circulating cell‐free DNA obtained from normal human subjects
title_full_unstemmed Whole genome sequencing of low input circulating cell‐free DNA obtained from normal human subjects
title_short Whole genome sequencing of low input circulating cell‐free DNA obtained from normal human subjects
title_sort whole genome sequencing of low input circulating cell‐free dna obtained from normal human subjects
topic Original Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8339531/
https://www.ncbi.nlm.nih.gov/pubmed/34350716
http://dx.doi.org/10.14814/phy2.14993
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