Design of Large‐Insert Jumping Libraries for Structural Variant Detection Using Illumina Sequencing

C. Hanscom1, M. Talkowski2

1 Molecular Neurogenetics Unit, Psychiatric and Neurodevelopmental Genetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, Massachusetts, 2 Department of Neurology, Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 7.22
DOI:  10.1002/0471142905.hg0722s80
Online Posting Date:  January, 2014
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Abstract

Next‐generation sequencing is an important and efficient tool for the identification of structural variation, particularly balanced chromosomal rearrangements, because such events are not routinely detected by microarray and localization of altered regions by karyotype is imprecise. Indeed, the degree of resolution that can be obtained through next‐generation technologies enables elucidation of precise breakpoints and has facilitated the discovery of numerous pathogenic loci in human disease and congenital anomalies. The protocol described here explains one type of large‐insert “jumping library” and the steps required to generate such a library for multiplexed sequencing using Illumina sequencing technology. This approach allows for cost‐efficient multiplexing of samples and provides a very high yield of fragments with large inserts, or “jumping” fragments. Curr. Protoc. Hum. Genet. 80:7.22.1‐7.22.9. © 2014 by John Wiley & Sons, Inc.

Keywords: next‐generation sequencing; mate pair sequencing; jumping libraries; structural variation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Generation of Libraries with Large Genomic Inserts for 25‐bp Paired‐End Illumina Platform Sequencing
  • Support Protocol 1: PCR Protocol for Library Amplification
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Generation of Libraries with Large Genomic Inserts for 25‐bp Paired‐End Illumina Platform Sequencing

  Materials
  • 5 to 10 µg genomic DNA sample (sample)
  • 1× TE buffer (Life Technologies, cat. no. 12090‐015)
  • QIAquick PCR Purification kit (cat. no. 28104 or cat. no. 28106)
  • End‐It DNA End‐Repair kit (Epicentre, cat. no. ER81050)
  • RNase‐Free Duplex buffer (IDT)
  • Cap adapter oligo duplex (see recipe)
  • Quick ligation kit (NEB, cat. no. M2200S or cat. no. M2200L) containing:
  •  Quick ligation buffer
  • UltraPure Agarose (Life Technologies, cat. no. 16500‐500)
  • Ethidium bromide (Sigma, cat. no. E1510‐10ML)
  • 6× loading dye
  • 1 kb+ DNA ladder (Life Technologies, cat. no. 10787‐018)
  • QIAquick Gel Extraction kit (cat. no. 28704 or cat. no. 28706)
  • Quant‐iT PicoGreen dsDNA Assay kit (Life Technologies, cat. no. P7589)
  • Internal circularization adapter duplex (see recipe)
  • Plasmid‐safe DNaSE kit (Epicentre, cat. no. E3105K)
  • EcoP15I restriction enzyme (NEB, cat. no. R0646L)
  • BSA, 100× (NEB, cat. no. B9000S)
  • 10 mM ATP (contained in NEB EcoP15I package; NEB)
  • NEB Buffer 3 (contained in NEB EcoP15I package; NEB)
  • Sinefungin, 10 mM (Millipore, cat. no. 567051‐2MG)
  • Klenow DNA polymerase I (lg) fragment (NEB, cat. no. M0210S)
  • 100 nM dNTP mix (Life Technologies, cat no. 10297‐018)
  • Ice
  • Streptavidin binding and wash buffers (see recipe)
  • Tween 20 (Sigma‐Aldrich, cat. no. P9416‐50ML)
  • Dynabeads MyOne Streptavidin C1 (Life Technologies, cat. no. 65001)
  • NEBNext dA Tailing Module (NEB, cat. no. E6053L)
  • Barcoded Y adapter duplex (see recipe)
  • Phusion High‐Fidelity PCR MM w/HF Buffer (NEB, cat. no. M0531S)
  • Agilent 1000 kit (Agilent, cat. no. 5067‐4626)
  • PhiX Control v2 Kit (Illumina, cat. no. 1006471)
  • Covaris Focused‐ultrasonicator (S‐Series or E‐Series; Covaris)
  • Covaris miniTUBES (blue) for 3 kb shearing (520065)
  • NanoDrop spectrophotometer (ND1000)
  • Gel electrophoresis apparatus (BIO‐RAD)
  • UV imaging apparatus for gel visualization
  • Protective face mask
  • Scalpels or razors for gel extraction
  • Heat block or incubator (37°C, 65°C; Eppendorf)
  • Vortex mixer
  • Centrifuge (Eppendorf, model no. 5804)
  • Magnetic rack (Invitrogen, cat. no. CS15000)
  • Thermal cycler (Bio‐Rad, model. no. C1000)
  • Agilent 2100 Bioanalyzer (Agilent 2100 or equivalent QC method)

Support Protocol 1: PCR Protocol for Library Amplification

  Materials
  • Barcode‐ligated DNA sample (on beads, resuspended in 30 µl EB; see the protocol 1Basic Protocol)
  • Univ_PCR forward primer (25 µM)
  • Index‐compatible PCR reverse primer (25 µM)
  • Phusion High‐Fidelity PCR MM w/HF buffer (NEB, cat. no. M0531S)
  • Nuclease‐free H 2O
  • Thermal cycler
  • Magnetic rack
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Figures

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Literature Cited

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Internet Resources
   http://tools.invitrogen.com/content/sfs/manuals/SOLiD4_Library_Preparation_man.pdf
   Web site for the Applied Biosystems SOLiD 4 System Library Preparation Guide, 2010.
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