Comparative analysis of 12 different kits for bisulfite conversion of circulating cell-free DNA

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Comparative analysis of 12 different kits for bisulfite conversion of circulating cell-free DNA. / Ørntoft, Mai-Britt Worm; Jensen, Sarah Østrup; Hansen, Thomas Birkballe; Bramsen, Jesper Bertram; Andersen, Claus Lindbjerg.

In: Epigenetics, Vol. 12, No. 8, 2017, p. 626-636.

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@article{ff1a63316cbc4b5a9fb030c2ceb63f3b,
title = "Comparative analysis of 12 different kits for bisulfite conversion of circulating cell-free DNA",
abstract = "Blood circulating cell-free DNA (cfDNA) is becoming popular in the search of promising predictive and prognostic biomarkers. Among these biomarkers, cfDNA methylation markers have especially gained considerable attention. A significant challenge in the utilization of cfDNA methylation markers is the limited amount of cfDNA available for analyses; reportedly, bisulfite conversion (BSC) reduce cfDNA amounts even further. Nevertheless, few efforts have focused on ensuring high cfDNA conversion efficiency and recovery after BSC. In order to compare cfDNA recovery of different BSC assays, we compared 12 different commercially available BSC kits. We tested whether DNA recovery was affected by the molecular weight and/or quantity of input DNA. We also tested BSC efficiency for each kit. We found that recovery varied for DNA fragments of different lengths: certain kits recovered short fragments better than others, and only three kits recovered DNA fragments of <100 bp well. In contrast, DNA input amount did not seem to affect DNA recovery: for quantities spanning between 820 and ∼25,000 genome equivalents per BSC, a linear relation was found between input and recovery amount. Overall, mean recovery ranged between 9 and 32%, with BSC efficiency of 97-99.9%. When plasma cfDNA was used as input for BSC, recovery varied from 22% for the poorest and 66% for the best performing kits, while conversion efficiency ranged from 96 to 100% among different kits. In conclusion, clear performance differences exist between commercially available BSC kits, both in terms of DNA recovery and conversion efficiency. The choice of BSC kit can substantially impact the amount of converted cfDNA available for downstream analysis, which is critical in a cfDNA methylation marker setting.",
keywords = "Journal Article",
author = "{\O}rntoft, {Mai-Britt Worm} and Jensen, {Sarah {\O}strup} and Hansen, {Thomas Birkballe} and Bramsen, {Jesper Bertram} and Andersen, {Claus Lindbjerg}",
year = "2017",
doi = "10.1080/15592294.2017.1334024",
language = "English",
volume = "12",
pages = "626--636",
journal = "Epigenetics",
issn = "1559-2294",
publisher = "Taylor & Francis Inc.",
number = "8",

}

RIS

TY - JOUR

T1 - Comparative analysis of 12 different kits for bisulfite conversion of circulating cell-free DNA

AU - Ørntoft, Mai-Britt Worm

AU - Jensen, Sarah Østrup

AU - Hansen, Thomas Birkballe

AU - Bramsen, Jesper Bertram

AU - Andersen, Claus Lindbjerg

PY - 2017

Y1 - 2017

N2 - Blood circulating cell-free DNA (cfDNA) is becoming popular in the search of promising predictive and prognostic biomarkers. Among these biomarkers, cfDNA methylation markers have especially gained considerable attention. A significant challenge in the utilization of cfDNA methylation markers is the limited amount of cfDNA available for analyses; reportedly, bisulfite conversion (BSC) reduce cfDNA amounts even further. Nevertheless, few efforts have focused on ensuring high cfDNA conversion efficiency and recovery after BSC. In order to compare cfDNA recovery of different BSC assays, we compared 12 different commercially available BSC kits. We tested whether DNA recovery was affected by the molecular weight and/or quantity of input DNA. We also tested BSC efficiency for each kit. We found that recovery varied for DNA fragments of different lengths: certain kits recovered short fragments better than others, and only three kits recovered DNA fragments of <100 bp well. In contrast, DNA input amount did not seem to affect DNA recovery: for quantities spanning between 820 and ∼25,000 genome equivalents per BSC, a linear relation was found between input and recovery amount. Overall, mean recovery ranged between 9 and 32%, with BSC efficiency of 97-99.9%. When plasma cfDNA was used as input for BSC, recovery varied from 22% for the poorest and 66% for the best performing kits, while conversion efficiency ranged from 96 to 100% among different kits. In conclusion, clear performance differences exist between commercially available BSC kits, both in terms of DNA recovery and conversion efficiency. The choice of BSC kit can substantially impact the amount of converted cfDNA available for downstream analysis, which is critical in a cfDNA methylation marker setting.

AB - Blood circulating cell-free DNA (cfDNA) is becoming popular in the search of promising predictive and prognostic biomarkers. Among these biomarkers, cfDNA methylation markers have especially gained considerable attention. A significant challenge in the utilization of cfDNA methylation markers is the limited amount of cfDNA available for analyses; reportedly, bisulfite conversion (BSC) reduce cfDNA amounts even further. Nevertheless, few efforts have focused on ensuring high cfDNA conversion efficiency and recovery after BSC. In order to compare cfDNA recovery of different BSC assays, we compared 12 different commercially available BSC kits. We tested whether DNA recovery was affected by the molecular weight and/or quantity of input DNA. We also tested BSC efficiency for each kit. We found that recovery varied for DNA fragments of different lengths: certain kits recovered short fragments better than others, and only three kits recovered DNA fragments of <100 bp well. In contrast, DNA input amount did not seem to affect DNA recovery: for quantities spanning between 820 and ∼25,000 genome equivalents per BSC, a linear relation was found between input and recovery amount. Overall, mean recovery ranged between 9 and 32%, with BSC efficiency of 97-99.9%. When plasma cfDNA was used as input for BSC, recovery varied from 22% for the poorest and 66% for the best performing kits, while conversion efficiency ranged from 96 to 100% among different kits. In conclusion, clear performance differences exist between commercially available BSC kits, both in terms of DNA recovery and conversion efficiency. The choice of BSC kit can substantially impact the amount of converted cfDNA available for downstream analysis, which is critical in a cfDNA methylation marker setting.

KW - Journal Article

U2 - 10.1080/15592294.2017.1334024

DO - 10.1080/15592294.2017.1334024

M3 - Journal article

C2 - 28557629

VL - 12

SP - 626

EP - 636

JO - Epigenetics

JF - Epigenetics

SN - 1559-2294

IS - 8

ER -