Next‐Generation Sequencing Library Preparation from FFPE Tissue Samples

Kathrin Wolf1, Dominic O'Neil1, Stefanie Schroeer1, Nan Fang1

1 Qiagen, Hilden
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 7.24
DOI:  10.1002/0471142727.mb0724s113
Online Posting Date:  January, 2016
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Abstract

Formalin‐fixed, paraffin‐embedded (FFPE) tissue samples represent an invaluable biobank for retrospective cancer research with molecular methods such as real‐time PCR and next‐generation sequencing (NGS). However, the usage of FFPE material in NGS approaches involves several challenges associated with the limited quantity and quality of DNA. This protocol describes how to purify DNA from FFPE material and how to prepare it for downstream NGS workflows including fragmentation, size selection, and library preparation for Illumina MiSeq/HiSeq sequencing. © 2016 by John Wiley & Sons, Inc.

Keywords: formalin‐fixed paraffin‐embedded tissue; next‐generation sequencing; fragmentation; library preparation; illumina

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Purification of DNA from FFPE Tissue Sections
  • Basic Protocol 2: Fragmentation of FFPE DNA
  • Basic Protocol 3: Preparation of a Sequencing Library for Paired‐End Sequencing
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Purification of DNA from FFPE Tissue Sections

  Materials
  • Freshly cut section of FFPE tissue, up to 10 μm
  • GeneRead DNA FFPE Kit (Qiagen, cat. no. 180134) containing:
    • Deparaffinization solution
    • RNase‐free water
    • Buffer FTB
    • Proteinase K
    • Uracil‐N‐glycosylase (UNG)
    • RNase A (100 mg/ml)
    • Buffer AL
    • QIAamp MinElute columns
    • 2‐ml collection tubes
    • Buffer AW1 (concentrate)
    • Buffer AW2 (concentrate)
    • Buffer ATE
  • 96% to 100% ethanol (nondenatured)
  • Scalpel
  • 1.5‐ml Safe‐Lock microcentrifuge tubes (Brinkmann, cat. no. 022363204 or Eppendorf, cat. no. 0030 120.086), or 1.5‐ml SafeSeal microcentrifuge tubes (Sarstedt, cat. no. 72.706), or 2‐ml Safe‐Lock microcentrifuge tubes (Brinkmann, cat. no. 022363352 or Eppendorf, cat. no. 0030 120.094), or 2‐ml SafeSeal microcentrifuge tubes (Sarstedt, cat. no. 72.695)
  • Vortex
  • Microcentrifuge with rotor for 2‐ml tubes
  • Thermal mixer or heating block capable of incubation at 90°C
CAUTION: Buffers AL and AW1 contain a chaotropic salt. Take appropriate laboratory safety measures and wear gloves when handling. Chaotropic salts are not compatible with disinfectants containing bleach and can become highly reactive compounds when combined with bleach.

Basic Protocol 2: Fragmentation of FFPE DNA

  Materials
  • Purified FFPE DNA of interest
  • Qubit dsDNA HS Assay Kit (Invitrogen, cat. no. Q32851)
  • Tris EDTA buffer, pH 8.0 (TE buffer; see appendix 22)
  • MinElute PCR Purification Kit (Qiagen, cat. No. 28004 or 28006) containing:
    • Buffer PB
    • Buffer PE
    • Buffer EB
    • QIAamp MinElute columns
    • 2‐ml collection tubes
  • Agilent High Sensitivity DNA Analysis Kit (Agilent, cat. No. 5067‐4626 or 5067‐4627)
  • S220 Focused‐ultrasonicator (Covaris)
  • microTUBE AFA Fiber Pre‐Slit Snap‐Cap 6 × 16–mm (Covaris, cat. no. 520045)
  • 1.5‐ml microcentrifuge tubes

Basic Protocol 3: Preparation of a Sequencing Library for Paired‐End Sequencing

  Materials
  • Purified FFPE DNA of interest
  • GeneRead NGS Library I Core Kit (Qiagen, cat. no. 180432) containing:
    • RNase‐free water
    • End‐repair buffer
    • End‐repair enzyme mix
    • A‐addition buffer
    • Klenow fragment
    • Ligation buffer
    • T4 DNA ligase
  • GeneRead Adapter I Set A/B 12‐plex (Qiagen, cat. nos. 180985 or 180986) or GeneRead Adapter I Set 1‐plex (Qiagen, cat. no. 180912)
  • GeneRead Size Selection Kit (for inserts >200 bp; Qiagen, cat. no. 180514) or Agencourt AMPure XP Kit (for inserts <200 bp; Beckman Coulter, cat. no. A63880)
  • GeneRead DNA I Amp Kit (Qiagen, cat. no. 180455) containing:
    • HiFi PCR master mix
    • Primer mix
  • MinElute PCR Purification Kit (for inserts <200 bp; Qiagen, cat. no. 28004)
  • Agilent High Sensitivity DNA Analysis Kit (Agilent, cat. no. 5067‐4626 or 5067‐4627)
  • GeneRead Library Quantification Kit (Qiagen, cat. no. 180612)
  • Ice
  • PCR tubes or PCR plate
  • Thermal cycler
  • Real‐time PCR instrument
NOTE: For NGS library preparation, use at least 100 ng of fragmented FFPE DNA and GeneRead DNA Library I Core Kit.
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Figures

Videos

Literature Cited

Literature Cited
  Do, H. and Dobrovic, A. 2012. Dramatic reduction of sequence artefacts from DNA isolated from formalin‐fixed cancer biopsies by treatment with uracil‐DNA glycosylase. Oncotarget 3:546‐558. doi: 10.18632/oncotarget.503.
  Klug, W.S. and Cummings, M.R. 1997. Concepts of Genetics, 5th ed. Prentice Hall, Upper Saddle River, N.J.
  Kokkat, T.J., Patel, M.S., McGarvey, D., LiVolsi, V.A., and Baloch, Z.W. 2013. Archived formalin‐fixed paraffin‐embedded (FFPE) blocks: A valuable underexploited resource for extraction of DNA, RNA, and protein. Biopreserv. Biobank. 11:101‐106. doi: 10.1089/bio.2012.0052.
  Ley, T.J., Mardis, E.R., Ding, L., Fulton, B., McLellan, M.D., Chen, K., Dooling, D., Dunford‐Shore, B.H., McGrath, S., Hickenbotham, M., Cook, L., Abbott, R., Larson, D.E., Koboldt, D.C., Pohl, C., Smith, S., Hawkins, A., Abbott, S., Locke, D., Hillier, L.W., Miner, T., Fulton, L., Magrini, V., Wylie, T., Glasscock, J., Conyers, J., Sander, N., Shi, X., Osborne, J.R., Minx, P., Gordon, D., Chinwalla, A., Zhao, Y., Ries, R.E., Payton, J.E., Westervelt, P., Tomasson, M.H., Watson, M., Baty, J., Ivanovich, J., Heath, S., Shannon, W.D., Nagarajan, R., Walter, M.J., Link, D.C., Graubert, T.A., DiPersio, J.F., and Wilson, R.K. 2008. DNA sequencing of a cytogenetically normal acute myeloid leukaemia genome. Nature 456:66‐72. doi: 10.1038/nature07485.
  Senguven, B., Baris, E., Oygur, T., and Berktas, M. 2014. Comparison of methods for the extraction of DNA from formalin‐fixed, paraffin‐embedded archival tissues. Int. J. Med. Sci. 11:494‐499. doi: 10.7150/ijms.8842.
  Srinivasan, M., Sedmak, D., and Jewell, S. 2002. Effect of fixatives and tissue processing on the content and integrity of nucleic acids. Am. J. Pathol. 161:1961‐1971. doi: 10.1016/S0002‐9440(10)64472‐0.
  Vollbrecht, C., Mairinger, F., Schweighofer, C., Heukamp, L., Merkelbach‐Bruse, S., Büttner, R., and Odenthal, M. 2014. Targeting cancer related genes by multiplex PCR followed by high throughput parallel sequencing. Int. J. Genomic Med. 2(1):115. doi:10.4172/2332‐0672.1000115
  Williams, C., Ponten, F., Moberg, C., Soderkvist, P., Uhlen, M., Ponten, J., Sitbon, G., and Lundeberg, J. 1999. A high frequency of sequence alterations is due to formalin fixation of archival specimens. Am. J. Pathol. 155:1467‐1471. doi: 10.1016/S0002‐9440(10)65461‐2.
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