Application Note: Reproductive and Genetic Health

Laboratory Experience Using Illumina
Next-Generation Sequencing for Noninvasive
Prenatal Testing
Noninvasive prenatal testing results generated on the NextSeq® 500 System are equivalent tothose achieved with the HiSeq® System.
Introduction

Experimental Studies

Prenatal screening provides pregnant women with the option ofidentifying particular fetal chromosomal abnormalities, including
Down syndrome (trisomy 21), Edwards syndrome (trisomy 18), Patau
syndrome (trisomy 13), and sex chromosome–related syndromes
such as Klinefelter and Turner syndromes. As shown in prior studies,noninvasive prenatal testing (NIPT) performed with next-generation
sequencing (NGS) offers a reliable, quick screen for chromosomal
abnormalities.1,2 NGS generates high-sensitivity, high-specificity NIPT
results with low false-positive and false-negative rates and maintains a
low test failure rate, minimizing the need for invasive testing procedures.3

The following 2 experiments demonstrate the potential use of theNextSeq 500 System for NIPT.

The verifi Prenatal Test* is an NGS-based noninvasive test that uses
the Illumina HiSeq System for sample sequencing. To demonstratethat the Illumina NextSeq 500 System can generate data equivalent
to that of the HiSeq System, 2 experiments sequencing a total of663 maternal samples for NIPT on both systems were performed.

Illumina NGS Systems
The Illumina HiSeq and NextSeq 500 NGS Sequencing Systems
provide the throughput, read length, and depth required for NIPT. TheHiSeq System is a massive-throughput sequencing instrument offering
power and efficiency for large-scale genomic studies. The NextSeq
500 System is a desktop instrument that provides a fast, integrated,sample-to-results workflow with push-button sequencing, simple data
analysis, and minimal hands-on time. The NextSeq 500 System delivers
the benefits of high-throughput sequencing at a lower capital expense
than the HiSeq System.

*The verifi Prenatal Test is a laboratory developed test and its performance characteristics
were determined by Verinata Health, Inc., a wholly owned subsidiary of Illumina, Inc. The
U.S. Food and Drug Administration has not approved or cleared this test.

Isolation
and Extraction

LibraryPreparation

Prepare cfDNA frommaternal blood

Prepare libraries forsequencing using
TruSeq DNASample Prep Kits

Experiment 1—DesignIn the first experiment, 551 maternal blood samples were preparedand sequenced on both the HiSeq and NextSeq 500 Systems
following the workflow outlined in Figure 1. Libraries for sequencing
were prepared using TruSeq® Nano DNA Library Prep Kits.
Sequencing on the NextSeq 500 and HiSeq Systems used the runparameters outlined in Table 1. Each instrument generated initial basecalls. Additional data processing was done using analysis methodsbased on the verifi Test analysis software.
Table 1: NGS Parameters for NIPT
HiSeq 2500
SystemNextSeq 500
SystemSamples per Run

1515

Reads Passing Filter

330 M

458 M

Read Length

36 bp

36 bp

Tags per Run

268 M

331 M

Experiment 1—Analysis and Results
Data generated for all 551 samples were compared across2 important NIPT metrics: ability to identify copy number changes
on chromosome X for male fetus samples and variation of signals
for unaffected (diploid) samples.
Copy number changes on chromosome X can be measured by relative
representation of chromosome X in the background of sequences fromother (“reference”) chromosomes, or chromosomal ratio X. Ratio X inSequencing
Start the sequencing
instrument
Add library to theready-to-use flow cell

DataAnalysis
Demultiplex samples
Align reads to genome

Generate
ReportAnalyze data for
aneuploidy
Generate report

Figure 1: NGS Workflow for NIPT—To demonstrate the performance of the verifi Prenatal Test on the NextSeq 500 System, experiments using both the NextSeq 500and HiSeq Systems followed the same workflow.

Application Note: Reproductive and Genetic Healthmale samples characterizes the response to fetal fraction and relates
directly to the ability of the system to detect aneuploidy. As shown in
Figure 2, the value of ratio X was similar when measured on HiSeq
and Next 500 Systems. This result indicates similar amplitude of copy
number variation in response to fetal fraction.
In addition to characterizing response to fetal fraction in male samples,
signal variation for unaffected samples on target chromosomes(13/18/21) was characterized by percent coefficient of variation (%CV)
of chromosomal ratios. Even though %CV is measured on unaffected
samples, it determines analytical sensitivity for detecting aneuploidies.
The %CV for chromosomes 13,18, and 21 was consistent between
platforms (Table 2), suggesting nearly identical assay performance
regarding detection of fetal aneuploidy for these chromosomes acrossthe HiSeq and NextSeq 500 Systems.

Experiment 2—Analysis and Results
Data obtained for the second set of 112 samples was used togenerate normalized chromosome 21 values (NCV 21, or the z-scores
used for NIPT classification). As shown in Figure 3 and predicted byprevious %CV analysis, the sequencing instrument used did not affectz-scores or the ability to detect aneuploidy in paired samples.	  

Figure 3: Nearly Identical NCV 21 Values—Plotting NCV 21 values of 112
samples sequenced using both the HiSeq and NextSeq 500 Systems showsnearly identical values, indicating the ability of both systems to determine thepresence of fetal trisomy 21 in NIPT. Grey: unaffected. Green: affected T21.

Summary	  Figure 2: High Concordance of Ratio X Values—The high level ofconcordance achieved when plotting the ratio X of each sample generatedon the NextSeq 500 and HiSeq Systems demonstrates that both produce thechromosomal ratios required for detection of fetal aneuploidy. Grey: male fetus.
Green: female fetus.

Table 2: Low, Consistent %CV Values Achieved With the HiSeq andNextSeq 500 Systems% CV HiSeq
2500 System% CV NextSeq
500 System

Chr. 130.197%

0.202%

Chr. 180.217%

0.200%

Chr. 210.294%

0.274%

Experiment 2—DesignA second sample set of 112 samples was sequenced on both theHiSeq and NextSeq 500 Systems. The second sample set contained9 samples that tested positive for fetal trisomy 21 by the verifi Prenatal
Test. Samples were prepared and sequenced according to theprotocols outlined in the “Experiment 1—Design” section.

This study assessed data relating to key NIPT metrics for 663 matchedsamples processed on both the HiSeq and NextSeq 500 Systems.
Results indicate that the NextSeq 500 System generates ratio X, %CV,and NCV 21 data that is comparable to the HiSeq System.

Learn More
To learn more about Illumina solutions for NIPT, visit www.illumina.
com/nextseqnote.

Reference1. Bianchi DW, Platt LD, Goldberg JD, Abuhamad AZ, Sehnert AJ,Rava RP. Genome-wide fetal aneuploidy detection by maternal
plasma DNA sequencing. Obstet Gynecol. 2012;119(5):890-901.
doi:10.1097/AOG.0b013e31824fb482.
2. Futch T, Spinosa J, Bhatt S, de Feo E, Rava RP, Sehnert AJ.
Initial clinical laboratory experience in noninvasive prenatal testingfor fetal aneuploidy from maternal plasma DNA samples. PrenatDiagn. 2013;33(6):569-574. doi:10.1002/pd.4123
3. Bianchi DW, Rava RP, Sehnert AJ. DNA sequencing versus
standard prenatal aneuploidy screening. N Engl J Med. 2014;
7;371(6):578. doi: 10.1056/NEJMc1405486.

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© 2015 Illumina, Inc. All rights reserved.
Illumina, HiSeq, NextSeq, TruSeq, verifi, and the pumpkin orange color are trademarks of Illumina, Inc. and/or its affiliate(s) in the U.S.
and/or other countries. Pub. No. 1570-2014-053 Current as of 23 March 2015