NEBNext Direct: A Novel, Rapid, Hybridization‐Based Approach for the Capture and Library Conversion of Genomic Regions of Interest

Amy B. Emerman1, Sarah K. Bowman1, Andrew Barry2, Noa Henig1, Kruti M. Patel1, Andrew F. Gardner2, Cynthia L. Hendrickson1

1 Directed Genomics, Inc., Ipswich, Massachusetts, 2 New England Biolabs, Ipswich, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 7.30
DOI:  10.1002/cpmb.39
Online Posting Date:  July, 2017
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Abstract

Next‐generation sequencing (NGS) is a powerful tool for genomic studies, translational research, and clinical diagnostics that enables the detection of single nucleotide polymorphisms, insertions and deletions, copy number variations, and other genetic variations. Target enrichment technologies improve the efficiency of NGS by only sequencing regions of interest, which reduces sequencing costs while increasing coverage of the selected targets. Here we present NEBNext Direct®, a hybridization‐based, target‐enrichment approach that addresses many of the shortcomings of traditional target‐enrichment methods. This approach features a simple, 7‐hr workflow that uses enzymatic removal of off‐target sequences to achieve a high specificity for regions of interest. Additionally, unique molecular identifiers are incorporated for the identification and filtering of PCR duplicates. The same protocol can be used across a wide range of input amounts, input types, and panel sizes, enabling NEBNext Direct to be broadly applicable across a wide variety of research and diagnostic needs. © 2017 by John Wiley & Sons, Inc.

Keywords: in‐solution hybridization; Illumina library preparation; next‐generation sequencing; NGS library preparation; NGS target capture; PCR enrichment; target enrichment

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Hybridization‐Based Method to Isolate Targets of Interest from Genomic DNA
  • Support Protocol 1: Analysis of Sequencing Reads
  • Alternate Protocol 1: NEBNext Direct Target Enrichment for Formalin‐Compromised DNA
  • Alternate Protocol 2: NEBNext Direct Target Enrichment for RNA Samples
  • Alternate Protocol 3: Overnight storage of Samples Mid‐Protocol
  • Commentary
  • Literarature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Hybridization‐Based Method to Isolate Targets of Interest from Genomic DNA

  Materials
  • 10 ng to 1 μg of input DNA
  • 1× TE buffer, pH 8 ( appendix 2A)
  • Agilent High Sensitivity DNA Kit (Agilent, cat. no. 5067‐4626)
  • NEBNext Direct Hybridization Buffer (NEB)
  • NEBNext Direct Hybridization Additive (NEB)
  • NEBNext Direct Baits (NEB)
  • NEBNext Direct Streptavidin Beads (NEB)
  • NEBNext Direct Hybridization Wash (HW; NEB)
  • NEBNext Direct Bead Prep Buffer (NEB)
  • NEBNext Direct Bead Wash 1 (BW1; NEB)
  • NEBNext Direct Bead Wash 2 (BW2; NEB)
  • NEBNext Direct 3′ Blunting Buffer (NEB)
  • NEBNext Direct 3′ Blunting Enzyme Mix (NEB)
  • NEBNext Direct dA‐Tailing Buffer (NEB)
  • NEBNext Direct dA‐Tailing Enzyme (NEB)
  • NEBNext Direct Adaptor Ligation Buffer (NEB)
  • NEBNext Direct 3′ Adaptor (NEB)
  • NEBNext Direct Ligase (NEB)
  • NEBNext Direct 5′ Blunting Buffer (NEB)
  • NEBNext Direct 5′ Blunting Enzyme Mix (NEB)
  • NEBNext Direct 5′ UMI Adaptor (NEB)
  • NEBNext Direct Cleaving Buffer (NEB)
  • NEBNext Direct Cleaving Enzyme Mix (NEB)
  • Molecular‐biology grade water
  • NEBNext Direct Q5 PCR Master Mix (NEB)
  • NEBNext Direct Index Primers (NEB)
  • NEBNext Direct Sample Purification Beads (NEB)
  • 80% (v/v) molecular‐biology‐grade ethanol (prepare fresh; use on same day)
  • Sterile, nuclease‐free microcentrifuge tubes
  • Covaris Focused‐ultrasonicator
  • Covaris microTubes or plate
  • Agilent Bioanalyzer or similar instrument
  • 96‐well PCR plates or PCR strip tubes
  • Eppendorf DNA LoBind 2‐ml tubes (VWR, cat. no. 80077‐234)
  • Thermocycler programmable to 100 μl
  • 96‐well plate magnet or PCR‐tube magnet for working with magnetic beads
  • Microcentrifuge tube magnet
  • Illumina MiSeq sequencing system
  • Additional reagents and equipment for PCR (Kramer & Coen, 2000)

Support Protocol 1: Analysis of Sequencing Reads

  Additional Materials (also see Basic Protocol 1)
  • 10 ng to 1 µg formalin‐compromised DNA
  • NEBNext Direct FFPE Phosphorylation Enzyme (NEB)
  • NEBNext Direct FFPE Phosphorylation Buffer (NEB)

Alternate Protocol 1: NEBNext Direct Target Enrichment for Formalin‐Compromised DNA

  Additional Materials (also see protocol 1Basic Protocol)
  • 20 ng to 1 µg total RNA
  • 5× NEBNext First Strand Synthesis Reaction Buffer
  • NEBNext Random Primers
  • Murine RNase inhibitor (NEB)
  • ProtoScript II Reverse Transcriptase (NEB)
  • 10× Second‐Strand Synthesis Reaction Buffer (NEB)
  • Second‐Strand Synthesis Enzyme Mix (NEB)
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Figures

Videos

Literature Cited

  Cibulskis, K., Lawrence, M. S., Carter, S. L., Sivachenko, A., Jaffe, D., Sougnez, C., … Getz, G. (2013). Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples. Nature Biotechnology, 31, 213–219. doi: 10.1038/nbt.2514.
  Forbes, S. A., Beare, D., Gunasekaran, P., Leung, K., Bindal, N., Boutselakis, H., … Campbell, P. J. (2015). COSMIC: Exploring the world's knowledge of somatic mutations in human cancer. Nucleic Acids Research, 43, D805–811. doi: 10.1093/nar/gku1075.
  Gnirke, A., Melnikov, A., Maguire, J., Rogov, P., LeProust, E. M., Brockman, W., … Nusbaum, C. (2009). Solution hybrid selection with ultra‐long oligonucleotides for massively parallel targeted sequencing. Nature Biotechnology, 27, 182–189. doi: 10.1038/nbt.1523.
  Hedegaard, J., Thorsen, K., Lund, M. K., Hein, A.‐M. K., Hamilton‐Dutoit, S. J., Vang, S., … Dyrskjøt, L. (2014). Next‐generation sequencing of RNA and DNA isolated from paired fresh‐frozen and formalin‐fixed paraffin‐embedded samples of human cancer and normal tissue. PLOS One, 9, e98187. doi: 10.1371/journal.pone.0098187.
  Johansson, H., Isaksson, M., Sörqvist, E. F., Roos, F., Stenberg, J., Sjöblom, T., … Nilsson, M. (2011). Targeted resequencing of candidate genes using selector probes. Nucleic Acids Research, 39, e8. doi: 10.1093/nar/gkq1005.
  Kozarewa, I., Armisen, J., Gardner, A. F., Slatko, B. E., & Hendrickson, C. L. (2015). Overview of target enrichment strategies. Current Protocols in Molecular Biology, 112, 7.21.1–7.21.23. doi: 10.1002/0471142727.mb0721s112.
  Kramer, M. F., & Coen, D. M. 2001. Enzymatic amplification of DNA by PCR: Standard procedures and optimization. Current Protocols in Molecular Biology, 56, 15.1:15.1.1–15.1.14. doi: 10.1002/0471142727.mb1501s56.
  Lai, Z., Markovets, A., Ahdesmaki, M., Chapman, B., Hofmann, O., McEwen, R., … Dry, J. R. (2016). VarDict: A novel and versatile variant caller for next‐generation sequencing in cancer research. Nucleic Acids Research, 44, e108. doi: 10.1093/nar/gkw227.
  Li, H., & Durbin, R. (2010). Fast and accurate long‐read alignment with Burrows‐Wheeler transform. Bioinformatics, 26, 589–595. doi: 10.1093/bioinformatics/btp698.
  Mertes, F., ElSharawy, A., Sauer, S., van Helvoort, J. M. L. M., van der Zaag, P. J., Franke, A., … Brookes, A. J. (2011). Targeted enrichment of genomic DNA regions for next‐generation sequencing. Briefings in Functional Genomics, 10, 374–386. doi: 10.1093/bfgp/elr033.
  Narzisi, G., O'Rawe, J. A., Iossifov, I., Fang, H., Lee, Y., Wang, Z., … Schatz, M. C. (2014). Accurate de novo and transmitted indel detection in exome‐capture data using microassembly. Nature Methods, 11, 1033–1036. doi: 10.1038/nmeth.3069.
  Porreca, G. J., Zhang, K., Li, J. B., Xie, B., Austin, D., Vassallo, S. L., … Shendure, J. (2007). Multiplex amplification of large sets of human exons. Nature Methods, 4, 931–936. doi: 10.1038/nmeth1110.
  Robinson, J. T., Thorvaldsdóttir, H., Winckler, W., Guttman, M., Lander, E. S., Getz, G., & Mesirov, J. P. (2011). Integrative genomics viewer. Nature Biotechnology, 29, 24–26. doi: 10.1038/nbt.1754.
  Shen, M.‐J. R., Oliphant, A., Butler, S. L., Stuelpnagel, J. E., Chee, M. S., Kuhn, K. M., & Fan, J.‐B. (2011). Multiplex nucleic acid reactions. U.S. Patent 7955794 B2.
  Sherry, S. T., Ward, M. H., Kholodov, M., Baker, J., Phan, L., Smigielski, E. M., & Sirotkin, K. (2001). dbSNP: The NCBI database of genetic variation. Nucleic Acids Research, 29, 308–311. doi: 10.1093/nar/29.1.308.
  Snyder, M. W., Kircher, M., Hill, A. J., Daza, R. M., & Shendure, J. (2016). Cell‐free DNA comprises an in vivo nucleosome footprint that informs its tissues‐of‐origin. Cell, 164, 57–68. doi: 10.1016/j.cell.2015.11.050.
  Srinivasan, M., Sedmak, D., & Jewell, S. (2002). Effect of fixatives and tissue processing on the content and integrity of nucleic acids. The American Journal of Pathology, 161, 1961–1971. doi: 10.1016/S0002‐9440(10)64472‐0.
  Tewhey, R., Warner, J. B., Nakano, M., Libby, B., Medkova, M., David, P. H., … Frazer, K. A. (2009). Microdroplet‐based PCR enrichment for large‐scale targeted sequencing. Nature Biotechnology, 27, 1025–1031. doi: 10.1038/nbt.1583.
  Thorvaldsdóttir, H., Robinson, J. T., & Mesirov, J. P. (2013). Integrative Genomics Viewer (IGV): High‐performance genomics data visualization and exploration. Briefings in Bioinformatics, 14, 178–192. doi: 10.1093/bib/bbs017.
  Varley, K. E., & Mitra, R. D. (2008). Nested Patch PCR enables highly multiplexed mutation discovery in candidate genes. Genome Research, 18, 1844–1850. doi: 10.1101/gr.078204.108.
Internet Resources
  https://www.neb.com/nebnext‐direct/nebnext‐direct‐for‐target‐enrichment
  NEBNext Direct Web site.
  https://www.neb.com/~/media/NebUs/Files/PDF%20FAQ/NEBNext%20Direct%20Cancer%20HotSpot%20Panel%20FAQ_4‐27.pdf
  NEBNext Direct FAQs: Includes additional information and resources including examples of command lines for data analysis.
  http://support.illumina.com/downloads/miseq_system_user_guide_15027617.html
  Illumina MiSeq System Guide.
  http://support.illumina.com/downloads/miseq‐reporter‐user‐guide‐15042295.html
  Illumina MiSeq Reporter Software Guide.
  http://support.illumina.com/downloads/prepare_libraries_for_sequencing_miseq_15039740.html
  MiSeq Denature and Dilute Libraries Guide.
  http://broadinstitute.github.io/picard
  Picard tools.
  https://software.broadinstitute.org/gatk/gatkdocs/
  Genome Analysis ToolKit (GATK).
  http://bio‐bwa.sourceforge.net/
  Burrows‐Wheeler Aligner (BWA) software package.
  https://github.com/fulcrumgenomics/fgbio
  Information on marking and working with duplicates.
  https://www.ncbi.nlm.nih.gov/snp
  The Single Nucleotide Polymorphism Database (dbSNP).
  http://cancer.sanger.ac.uk
  Cosmic database.
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