Construction of a Sequencing Library from Circulating Cell‐Free DNA

Nan Fang1, Dirk Löffert2, Rumeysa Akinci‐Tolun1, Katja Heitz1, Alexander Wolf1

1 QIAGEN GmbH, Hilden, 2 BIOMILLENIA, Paris
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 7.25
DOI:  10.1002/0471142727.mb0725s114
Online Posting Date:  April, 2016
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Abstract

Circulating DNA is cell‐free DNA (cfDNA) in serum or plasma that can be used for non‐invasive prenatal testing, as well as cancer diagnosis, prognosis, and stratification. High‐throughput sequence analysis of the cfDNA with next‐generation sequencing technologies has proven to be a highly sensitive and specific method in detecting and characterizing mutations in cancer and other diseases, as well as aneuploidy during pregnancy. This unit describes detailed procedures to extract circulating cfDNA from human serum and plasma and generate sequencing libraries from a wide concentration range of circulating DNA. © 2016 by John Wiley & Sons, Inc.

Keywords: circulating DNA; cfDNA; NGS; sequencing library preparation; low input

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Extraction of Circulating DNA from Human Blood or Serum
  • Basic Protocol 2: Construction of Sequencing Library from Circulating DNA Using Silica‐Column‐Based Reaction Clean‐Up
  • Alternate Protocol 1: Construction of Sequencing Library from Circulating DNA Using Agencourt Ampure XP Beads for Reaction Clean‐Up
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Extraction of Circulating DNA from Human Blood or Serum

  Materials
  • QIAamp Circulating Nucleic Acid Kit (QIAGEN, cat. no. 55114)
  • 96% to 100% ethanol
  • 100% isopropanol
  • Phosphate‐buffered saline (PBS; appendix 22)
  • Serum or plasma sample containing circulating DNA
  • 60°C water bath
  • 56°C heat block
  • Vacuum manifold (e.g., the QIAvac 24 Plus with the QIAvac Connecting System, cat. no. 19413)
  • Vacuum pump capable of producing a vacuum of –800 to –900 mbar (e.g., QIAGEN, cat. no. 19419)
  • 50‐ml centrifuge tubes

Basic Protocol 2: Construction of Sequencing Library from Circulating DNA Using Silica‐Column‐Based Reaction Clean‐Up

  Materials
  • Circulating cfDNA, extracted with QIAamp Circulating Nucleic Acid Kit ( protocol 1)
  • GeneRead DNA Library I Core Kit (QIAGEN, cat. no. 180432 or 180434)
  • GeneRead Adapters (QIAGEN, cat. no. 180985 or 180986)
  • GeneRead Size Selection Kit (QIAGEN, cat. no. 180514)
  • 80% ethanol
  • GeneRead DNA I Amp Kit (QIAGEN, cat. no. 180455)
  • PCR tubes or plates
  • Thermal cycler
  • Capillary electrophoresis device (e.g., 2100 Bioanalyzer from Agilent)
  • DNA LoBind tubes (from Eppendorf)
  • Additional reagents and equipment for the polymerase chain reaction (PCR; unit 15.1; Kramer and Coen, )

Alternate Protocol 1: Construction of Sequencing Library from Circulating DNA Using Agencourt Ampure XP Beads for Reaction Clean‐Up

  Materials
  • GeneRead DNA Library I Core Kit (QIAGEN, cat. no. 180432 or 180434)
  • GeneRead Adapters (QIAGEN, cat. no. 180985 or 180986)
  • Circulating cfDNA, extracted with QIAamp Circulating Nucleic Acid Kit ( protocol 1)
  • Agencourt AMPure XP Beads (Beckman Coulter, cat. no. A63880)
  • 80% ethanol
  • GeneRead DNA I Amp Kit (QIAGEN, cat. no. 180455)
  • GeneRead Library Quant Kit (QIAGEN, cat. no. 180612)
  • PCR tubes or plates
  • Thermal cycler
  • Magnetic racks for magnetic bead separation (e.g., DynaMag‐2 Magnet; Thermo Fisher Scientific/Life Technologies, cat.no. 12321D)
  • DNA LoBind tubes (from Eppendorf)
  • Additional reagents and equipment for the polymerase chain reaction (PCR; unit 15.1; Kramer and Coen, )
NOTE: 10 μl of circulating cfDNA extracted with the QIAamp Circulating Nucleic Acid Kit is used for next‐generation sequencing (NGS) library construction.
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Figures

Videos

Literature Cited

Literature Cited
  Aird, D., Ross, M.G., Chen, W.S., Danielsson, M., Fennell, T., Russ, C., Jaffe, D.B., Nusbaum, C., and Gnirke, A. 2011. Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries. Genome Biol. 12:R18. doi: 10.1186/gb-2011-12-2-r18.
  Crowley, E., Di Nicolantonio, F., Loupakis, F., and Bardelli, A. 2013. Liquid biopsy: Monitoring cancer‐genetics in the blood. Nat. Rev. Clin. Oncol. 10:472‐484. doi: 10.1038/nrclinonc.2013.110.
  Elshimali, Y.I., Khaddour, H., Sarkissyan, M., Wu, Y., and Vadgama, J.V. 2013. The clinical utilization of circulating cell free DNA (CCFDNA) in blood of cancer patients. Int. J. Mol. Sci. 14:18925‐18958. doi: 10.3390/ijms140918925.
  Hill, M., Wright, D., Daley, R., Lewis, C., McKay, F., Mason, S., Lench, N., Howarth, A., Boustred, C., Lo, K., Plagnol, V., Spencer, K., Fisher, J., Kroese, M., Morris, S., and Chitty, L.S. 2014. Evaluation of non‐invasive prenatal testing (NIPT) for aneuploidy in an NHS setting: A reliable accurate prenatal non‐invasive diagnosis (RAPID) protocol. BMC Pregnancy Childbirth 14:229. doi: 10.1186/1471-2393-14-229.
  Kramer, M.F. and Coen, D.M. 2000. Enzymatic amplification of DNA by PCR: Standard procedures and optimization. Curr. Protoc. Mol. Biol. 56:15.1.1‐15.1.14.
  Mandel, P. and Metais, P. 1948. Les acides nucleiques du plasma sanguin chez l'homme. CR Acad. Sci. Paris 142:241‐243.
  Pinzani, P., Salvianti, F., Pazzagli, M., and Orlando, C. 2010. Circulating nucleic acids in cancer and pregnancy. Methods 50:302‐307. doi: 10.1016/j.ymeth.2010.02.004.
  Quail, M.A., Otto, T.D., Gu, Y., Harris, S.R., Skelly, T.F., McQuillan, J.A., Swerdlow, H.P., and Oyola, S.O. 2012. Optimal enzymes for amplifying sequencing libraries. Nat. Methods 9:10‐11. doi: 10.1038/nmeth.1814.
  Song, K., Musci, T. J., and Caughey, A.B., 2013. Clinical utility and cost of non‐invasive prenatal testing with cfDNA analysis in high‐risk women based on a US population. J. Matern. Fetal Neonatal Med. 26:1180‐1185. doi: 10.3109/14767058.2013.770464.
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