Use of Spiked Normalizers to More Precisely Quantify Tumor Markers and Viral Genomes by Massive Parallel Sequencing of Plasma DNA
Copyright © 2020 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved..
A problematic aspect of massive parallel sequencing is that somatic mutations and viral loads are typically quantified as a fraction relative to wild-type human DNA, yet wild-type levels vary with diverse biologic and preanalytic interferences. A novel strategy was devised to quantify target analytes in copies per mL of plasma after normalizing for read counts of spiked DNAs. Five synthetic DNAs (called EndoGenus spikes) were added to plasma before library preparation (modified ArcherDX LiquidPlex 28). By normalizing to the fractional recovery of EndoGenus spike reads, numerical values for each disease marker were reportable in units of copies per mL. To show how well this system operates, replicate assays were performed on 40 mock plasmas having 23 engineered mutations and on 21 natural plasmas. Reads for all five EndoGenus spikes were recovered (means, 313 and 376 copies/mL in mock and natural plasmas, respectively). Normalizing read counts for the proportional recovery of spikes helped control for variables in the multistep protocol, reducing the CV in replicate tests from 34% to 22% for mutations and from 25% to 7% for viral loads. In conclusion, the EndoGenus system is useful for evaluating efficiency of the total test system and for precisely quantifying target molecules. This system may benefit patients being monitored for disease burden while also tracking emerging subclones.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2020 |
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| Erschienen: |
2020 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:22 |
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| Enthalten in: |
The Journal of molecular diagnostics : JMD - 22(2020), 4 vom: 15. Apr., Seite 437-446 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Gulley, Margaret L [VerfasserIn] |
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| Links: |
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| Themen: |
Biomarkers, Tumor |
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| Anmerkungen: |
Date Completed 04.06.2021 Date Revised 04.06.2021 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1016/j.jmoldx.2020.01.012 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM306306670 |
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| 245 | 1 | 0 | |a Use of Spiked Normalizers to More Precisely Quantify Tumor Markers and Viral Genomes by Massive Parallel Sequencing of Plasma DNA |
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| 500 | |a Date Revised 04.06.2021 | ||
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| 500 | |a Citation Status MEDLINE | ||
| 520 | |a Copyright © 2020 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. | ||
| 520 | |a A problematic aspect of massive parallel sequencing is that somatic mutations and viral loads are typically quantified as a fraction relative to wild-type human DNA, yet wild-type levels vary with diverse biologic and preanalytic interferences. A novel strategy was devised to quantify target analytes in copies per mL of plasma after normalizing for read counts of spiked DNAs. Five synthetic DNAs (called EndoGenus spikes) were added to plasma before library preparation (modified ArcherDX LiquidPlex 28). By normalizing to the fractional recovery of EndoGenus spike reads, numerical values for each disease marker were reportable in units of copies per mL. To show how well this system operates, replicate assays were performed on 40 mock plasmas having 23 engineered mutations and on 21 natural plasmas. Reads for all five EndoGenus spikes were recovered (means, 313 and 376 copies/mL in mock and natural plasmas, respectively). Normalizing read counts for the proportional recovery of spikes helped control for variables in the multistep protocol, reducing the CV in replicate tests from 34% to 22% for mutations and from 25% to 7% for viral loads. In conclusion, the EndoGenus system is useful for evaluating efficiency of the total test system and for precisely quantifying target molecules. This system may benefit patients being monitored for disease burden while also tracking emerging subclones | ||
| 650 | 4 | |a Journal Article | |
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| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
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| 650 | 7 | |a Cell-Free Nucleic Acids |2 NLM | |
| 650 | 7 | |a Circulating Tumor DNA |2 NLM | |
| 650 | 7 | |a DNA, Neoplasm |2 NLM | |
| 650 | 7 | |a DNA, Viral |2 NLM | |
| 700 | 1 | |a Elmore, Sandra |e verfasserin |4 aut | |
| 700 | 1 | |a Gupta, Gaorav P |e verfasserin |4 aut | |
| 700 | 1 | |a Kumar, Sunil |e verfasserin |4 aut | |
| 700 | 1 | |a Egleston, Matthew |e verfasserin |4 aut | |
| 700 | 1 | |a Hoskins, Ian J |e verfasserin |4 aut | |
| 700 | 1 | |a Garnett, Aaron |e verfasserin |4 aut | |
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