Balanced Chromosomal Rearrangement Detection by Low‐Pass Whole‐Genome Sequencing

Zirui Dong1, Lingfei Ye2, Zhenjun Yang3, Haixiao Chen2, Jianying Yuan2, Huilin Wang4, Xiaosen Guo2, Yun Li2, Jun Wang2, Fang Chen2, Sau Wai Cheung5, Cynthia C. Morton6, Hui Jiang2, Kwong Wai Choy7

1 BGI‐Shenzhen, Shenzhen, 2 China National Genebank‐Shenzhen, BGI‐Shenzhen, Shenzhen, 3 School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 4 Department of Central Laboratory, Bao'an Maternity and Child Healthcare Hospital, Jinan University School of Medicine, Key Laboratory of Birth Defects Research, Birth Defects Prevention Research and Transformation Team, Shenzhen, 5 Department of Molecular and Human Genetics, Baylor College of Medicine Houston, Texas, 6 Division of Evolution and Genomic Sciences, School of Biological Sciences, University of Manchester, Manchester Academic Health Science Center, Manchester, 7 The Chinese University of Hong Kong‐Baylor College of Medicine Joint Center for Medical Genetics
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 8.18
DOI:  10.1002/cphg.51
Online Posting Date:  January, 2018
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Balanced chromosomal rearrangements (or balanced chromosome abnormalities, BCAs) are common chromosomal structural variants. Emerging studies have demonstrated the feasibility of using whole‐genome sequencing (WGS) for detection of BCA‐associated breakpoints, but the requirement for a priori knowledge of the rearranged regions from G‐banded chromosome analysis limits its application. The protocols described here are based on low‐pass WGS for detecting BCA events independent from chromosome analysis, and has been validated using genomic data from the 1000 Genomes Project. This approach adopts non‐size‐selected mate‐pair library (3∼8 kb) with 2∼3 μg DNA as input, and requires only 30 million read‐pairs (50 bp, equivalent to 1‐fold base‐coverage) for each sample. The complete procedure takes 13 days and the total cost is estimated to be less than $600 (USD) per sample. © 2018 by John Wiley & Sons, Inc.

Keywords: balanced translocations; chromosomal structural rearrangements; low‐pass whole‐genome sequencing; inversions

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Table of Contents

  • Introduction
  • Basic Protocol 1: DNA Fragmentation and Library Construction
  • Basic Protocol 2: PCR Amplification, Size Selection, and Measurement
  • Basic Protocol 3: Bioinformatics Pipeline
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: DNA Fragmentation and Library Construction

  • DNA sample
  • DL2000 marker (Tiangen Biotech)
  • λ‐HindIII marker (Takara Biotechnology)
  • SYBR Safe DNA gel stain (Thermo Fisher Scientific)
  • Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific)
  • 1× TE buffer, pH 8.0 (Ambion, Thermo Fisher Scientific)
  • 0.2 M HCl (Ambion)
  • 0.2 M NaOH (Ambion)
  • Milli‐Q water
  • Anhydrous ethanol
  • 1‐kb DNA ladder (Tiangen)
  • T4 polynucleotide kinase (PNK) with 10× buffer (Enzymatics)
  • T4 DNA polymerase (Enzymatics)
  • Klenow DNA polymerase (Enzymatics)
  • dNTP mix, 25 mM (Invitrogen, Thermo Fisher Scientific)
  • Agencourt AMPure beads (Beckman Coulter, cat. no. A29152)
  • EB Buffer (Qiagen)
  • Biotin‐dNTP mix (Enzymatics, Qiagen)
  • 10× T4 DNA ligase with 10× buffer (Enzymatics)
  • 25 mM ATP (Enzymatics)
  • Plasmid‐safe DNase with 10× buffer (Epicentre Biotechnologies)
  • Exonuclease I (New England Biolabs)
  • 0.5 M EDTA (Ambion)
  • QIAquick PCR Purification Kit (Qiagen)
  • Dynabeads M‐280 Streptavidin (Invitrogen, Thermo Fisher Scientific)
  • 10× Blue Buffer (Enzymatics)
  • 1 mM dATP (Enzymatics)
  • Klenow 3′‐5′ (exo) (Enzymatics)
  • 2× Rapid Buffer (Enzymatics)
  • 2‐μm dry borosilicate glass microspheres (Duke Scientific, Thermo Fisher Scientific, cat. no. 9002)
  • PE Index Adapter Oligo Mix (Invitrogen)
  • Bead binding buffer (see recipe)
  • Bead wash buffer (see recipe)
  • NanoDrop 2000 (Thermo Fisher Scientific)
  • Qubit fluorometer (Thermo Fisher Scientific)
  • ThermoMixer (Eppendorf)
  • 1.5‐ml microcentrifuge tubes
  • 2‐μm filter needles with syringes (BD Biosciences)
  • 50‐ml tubes (BD Biosciences)
  • HydroShear shearing device (GeneMachines) with large custom shearing assembly (4‐40 kb) and 500‐μl syringe
  • Magnetic separator (Dexter Magnetic Technologies)
  • Covaris S2 ultrasonicator with 13 × 65–mm vials (Covaris)
  • 1.5‐ml non‐stick RNase‐free microcentrifuge tubes (Ambion)
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7; Jarcho, )

Basic Protocol 2: PCR Amplification, Size Selection, and Measurement

  • DNA library (see protocol 1)
  • Phusion DNA polymerase (New England Biolabs)
  • PCR primers 1.0 and 2.0 (see Table 8.17.1 in unit 8.17)
  • Milli‐Q water
  • 50‐bp DNA ladder (New England Biolabs)
  • QIAquick PCR Purification Kit (Qiagen)
  • EB Buffer (Qiagen)
  • ThermoMixer (Eppendorf)
  • 1.5 ml non‐stick RNase‐free microcentrifuge tubes (Ambion)
  • Magnetic separator (Dexter Magnetic Technologies)
  • Bioanalyzer 2100 (Agilent)
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7; Jarcho, ) and for assessing and pooling libraries (unit 8.17)

Basic Protocol 3: Bioinformatics Pipeline

  • Alignment file (via Burrows‐Wheeler aligner [bwa]; Li & Durbin, ): BCA.tar.gz
  • Linux‐based command system
  • SAMtools ( and Picard (
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Literature Cited

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