Noninvasive Prenatal Testing Using Cell‐Free Fetal DNA in Maternal Plasma

Nilesh Dharajiya1, Tricia Zwiefelhofer2, Xiaojun Guan3, Vach Angkachatchai2, Juan‐Sebastian Saldivar1

1 Sequenom Laboratories, Clinical Lab, San Diego, California, 2 Sequenom Laboratories, Diagnostic Development, San Diego, California, 3 Sequenom Laboratories, Bioinformatics, San Diego, California
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 8.15
DOI:  10.1002/0471142905.hg0815s84
Online Posting Date:  January, 2015
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Abstract

Noninvasive prenatal testing (NIPT) represents an outstanding example of how novel scientific discoveries can be quickly and successfully developed into hugely impactful clinical diagnostic tests. Since the introduction of NIPT to detect trisomy 21 in late 2011, the technology has rapidly advanced to analyze other autosomal and sex chromosome aneuploidies, and now includes the detection of subchromosomal deletion and duplication events. Here we provide a brief overview of how noninvasive prenatal testing using next‐generation sequencing is performed. © 2015 by John Wiley & Sons, Inc.

Keywords: Noninvasive prenatal testing; Next Generation Sequencing

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Sample Processing and Plasma Separation
  • Basic Protocol 2: Extraction of DNA from Plasma
  • Basic Protocol 3: Library Preparation
  • Basic Protocol 4: Library Quantitation and Normalization
  • Basic Protocol 5: Multiplexing and Clustering on the Illumina cBOT Instrument
  • Basic Protocol 6: Sequencing on the Illumina HiSeq2000 Instrument
  • Basic Protocol 7: Sequence Data Analysis
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Sample Processing and Plasma Separation

  Materials
  • Whole‐blood samples taken in Streck cell‐free DNA blood collection tubes (http://www.streck.com/)
  • Centrifuge with swinging‐bucket rotor
  • 15‐ml conical tubes (e.g., BD Falcon)

Basic Protocol 2: Extraction of DNA from Plasma

  Materials
  • QIAamp DSP Circulating Nucleic Acid kit (Qiagen, for in vitro Diagnostics) containing:
    • Lyophilized carrier RNA (310 μg)
    • Qiagen Proteinase K (4 × 7.0 ml)
    • Buffer ACL (lysis buffer; 220 ml)
    • Buffer ACB concentrate (binding buffer; 300 ml)
    • Buffer ACW1 concentrate (wash buffer 1; 19 ml)
    • Buffer ACW2 concentrate (wash buffer 2; 13 ml)
    • Buffer AVE (elution buffer; 5 × 2 ml)
    • QIAamp Mini Columns (50)
    • VacConnectors (50)
    • Tube or column extenders (25 × 2)
    • Elution Tubes (50 × 1.5 ml)
    • Wash or collection tubes (50)
  • Isopropanol for reconstitution of buffers
  • Absolute ethanol for reconstitution of buffers and washes
  • Patient plasma samples for analysis ( protocol 1)
  • 1× phosphate buffered saline (PBS) pH 7.2 ( appendix 2D), to make up plasma volume to 4 ml
  • Water bath capable of 60°C
  • Heat block capable of 56°C
  • Refrigerated centrifuge
  • 50‐ml conical centrifuge tubes (e.g., BD Falcon)
  • QIAvac 24 Plus Manifold or similar (Qiagen)
  • QIAvac Connecting System with regulated vacuum control or similar device (Qiagen)
  • Vacuum pump
  • VacValves (Qiagen, cat. no. 19408)
  • Microcentrifuge capable of 20,000 × g
NOTE: Buffers ACL, ACB, and ACW1 contain chemicals that react with bleach, forming dangerous vapors. If spills occur, clean up well with non‐bleach laboratory detergent and water.

Basic Protocol 3: Library Preparation

  Materials
  • Agencourt AMPure XP PCR Purification system (Beckman Coulter)
  • 200‐proof ethanol (molecular biology grade)
  • HPLC‐grade H 2O
  • NEBNext Ultra DNA Library Prep Kit for Illumina including:
    • NEBNext End Prep Enzyme Mix
    • 10× NEBNext End Repair Reaction Buffer
    • Blunt/TA Ligase Master Mix
    • NEBNext Ligation Enhancer
    • NEBNext High‐Fidelity 2× PCR Master Mix
  • NEBNext Multiplex Oligos for Illumina including:
    • NEBNext Adaptor for Illumina
    • USER Enzyme
    • Index Primers Set 1 or 2
  • cfDNA samples in an Eppendorf 96‐well full‐skirted reaction plate (∼50 μl per sample)
  • Buffer EB (Qiagen)
  • End‐over‐end rotator
  • Multi‐channel pipettor (e.g., Rainin), optional
  • Centrifuge with microtiter plate adaptor
  • Adhesive plate sealers for 96‐well plates
  • Thermal cycler: Eppendorf Mastercycler Pro S
  • Magnet plate (V&P Scientific, P/N VP771LD‐5CS)
  • Eppendorf 96‐well full‐skirted PCR reaction plate

Basic Protocol 4: Library Quantitation and Normalization

  Materials
  • DNA High Sensitivity Reagent Kit (PerkinElmer, part no. CLS760672) including:
    • Dye Concentrate
    • Gel Matrix
    • Ladder
    • HT DNA HiSens Marker
    • V‐bottom Buffer Tubes
    • 0.2‐ml Ladder Tubes
  • Isopropanol (molecular biology grade)
  • HPLC‐grade H 2O
  • Buffer EB (Qiagen)
  • Stock libraries ( protocol 3)
  • Centrifuge with microtiter plate adaptor
  • Vacuum filter flask setup
  • Eppendorf 96‐well full‐skirted PCR reaction plates
  • DNA Extended Range LabChip (PerkinElmer, part no. 760517)
  • Clean‐room cloth
  • LabChip GX Touch HT (PerkinElmer, part no. CLS137031)

Basic Protocol 5: Multiplexing and Clustering on the Illumina cBOT Instrument

  Materials
  • Illumina TruSeq SR Cluster Kit v3‐cBOT‐HS, including:
    • Single‐Read HiSeq v3 Flowcell (store at 4°C )
    • Single‐Read Cluster Kit (store at −20°C ) including Single‐Read cBOT Reagent Plate and HT1 Buffer
    • Hybridization Manifold for Flowcell v3 (store at room temperature)
    • Accessory Kit (store at room temperature)
    • Multiplex Sequencing Primer Kit (store at −20°C )
  • Buffer EB (Qiagen)
  • Normalized libraries ( protocol 4)
  • 2 N NaOH (molecular biology grade)
  • HPLC‐grade water
  • Illumina cBOT instrument
  • Centrifuge with microtiter plate carrier
  • 8‐well tube strip (VWR, cat. no. 20170‐004)

Basic Protocol 6: Sequencing on the Illumina HiSeq2000 Instrument

  Materials
  • Illumina TruSeq SBS Kit v3‐HS (50 cycle) including:
    • Box 1 of 2 (store at 4°C) with:
    • PW1 (wash buffer; quantity, 1)
    • ICB (Incorporation Mix Buffer; quantity, 1)
    • SB1 (High Salt Buffer; quantity, 1)
    • SB2 (Incorporation Wash Buffer; quantity, 2)
    • SB3 (Cleavage Buffer; quantity, 1)
    • Box 2 of 2 (store at −20°C) with:
    • CMR (Cleavage Mix Reagent; quantity, 1)
    • SRE (Enhanced Scan Mix Reagent; quantity, 1)
    • EDP (Enhanced DNA Polymerase; quantity, 1)
    • LFN36 (Long Read Nucleotide Mix; quantity, 1)
  • Multiplexing Sequencing Primer Kit (from the Illumina TruSeq SR Cluster Kit v3‐cBOT‐HS (see protocol 5) including:
    • HP8 (Index Sequencing Primer; quantity, 1)
    • HP3 (Denaturation Solution; quantity, 1)
    • HT2 (Wash Buffer; quantity, 1)
  • Flowcell with multiplexed, clustered samples ( protocol 5)
  • Ethanol, molecular biology grade
  • 15‐ml conical centrifuge tubes (e.g., BD Falcon)
  • Illumina HiSeq2000 sequencer w/HCS v1.4/RTA v1.12, or later
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Figures

Videos

Literature Cited

Literature Cited
  American College of Obstetricians and Gynecologists Committee on Genetics 2012. Committee opinion no. 545: Noninvasive prenatal testing for fetal aneuploidy. Obstet. Gynecol. 120:1532‐1534.
  Bianchi, D.W., Parker, R.L., Wentworth, J., Madankumar, R., Saffer, C., Das, A.F., Craig, J.A., Chudova, D.I., Devers, P.L., Jones, K.W., Oliver, K., Rava, R.P., Sehnert, A.J.; CARE Study Group. 2014. DNA sequencing versus standard prenatal aneuploidy screening. N. Engl. J. Med. 370:799‐808.
  Carraro, P. and Plebani, M. 2007. Errors in a stat laboratory: Types and frequencies 10 years later. Clin. Chem. 53:1338‐1342.
  Langmead, B. and Salzberg, S.L. 2012. Fast gapped‐read alignment with Bowtie 2. Nat. Methods 9:357‐359.
  Lo, Y.M., Corbetta, N., Chamberlain, P.F., Rai, V., Sargent, I.L., Redman, C.W., and Wainscoat, J.S. 1997. Presence of fetal DNA in maternal plasma and serum. Lancet 350:485‐487.
  Malone, F.D., Canick, J.A., Ball, R.H., Nyberg, D.A., Comstock, C.H., Bukowski, R., Berkowitz, R.L., Gross, S.J., Dugoff, L., Craigo, S.D., Timor‐Tritsch, I.E., Carr, S.R., Wolfe, H.M., Dukes, K., Bianchi, D.W., Rudnicka, A.R., Hackshaw, A.K., Lambert‐Messerlian, G., Wald, N.J., D'Alton, M.E.; First‐ and Second‐Trimester Evaluation of Risk (FASTER) Research Consortium. 2005. First‐trimester or second‐trimester screening, or both, for Down's syndrome. N. Engl. J. Med. 353:2001‐2011.
  Mazloom, A.R., Džakula, Ž., Oeth, P., Wang, H., Jensen, T., Tynan, J., McCullough, R., Saldivar, J.‐S., Ehrich, M., van den Boom, D., Bombard, A.T., Maeder, M., McLennan, G., Meschino, W., Palomaki, G.E., Canick, J.A., and Deciu, C. 2013. Noninvasive prenatal detection of sex chromosomal aneuploidies by sequencing circulating cell‐free DNA from maternal plasma. Prenatal Diagn. 33:591‐597.
  Palomaki, G.E., Kloza, E.M., Lambert‐Messerlian, G.M., Haddow, J.E., Neveux, L.M., Ehrich, M., van den Boom, D., Bombard, A.T., Deciu, C., Grody, W.W., Nelson, S.F., and Canick, J.A. 2011. DNA sequencing of maternal plasma to detect Down syndrome: An international clinical validation study. Genet. Med. 13:913‐920.
  Palomaki, G.E., Deciu, C., Kloza, E.M., Lambert‐Messerlian, G.M., Haddow, J.E., Neveux, L.M., Ehrich, M., van den Boom, D., Bombard, A.T., Grody, W.W., Nelson, S.F., and Canick, J.A. 2012. DNA sequencing of maternal plasma reliably identifies trisomy 18 and trisomy 13 as well as Down syndrome: An international collaborative study. Genet. Med 14:296‐305.
  Tabor, A., Philip, J., Madsen, M., Bang, J., Obel, E.B., and Nørgaard‐Pedersen, B. 1986. Randomised controlled trial of genetic amniocentesis in 4606 low‐risk women. Lancet 1:1287‐1293.
  Wians, F.H. 2009. Clinical Laboratory Tests: Which, Why, and What Do The Results Mean? LabMedicine 40:105‐113. Available at: http://labmed.ascpjournals.org/content/40/2/105.full [Accessed June 3, 2014].
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