VeriSeq NIPT Solution v2
Package Insert
FOR IN VITRO DIAGNOSTIC USEIntended Use
The VeriSeq™ NIPT Solution v2 is an in vitro diagnostic test intended for use as a screening test for thedetection of genome-wide fetal genetic anomalies from maternal peripheral whole blood specimens in pregnant
women of at least 10 weeks gestation. VeriSeq NIPT Solution v2 uses whole genome sequencing to detect
partial duplications and deletions for all autosomes and aneuploidy status for all chromosomes. The test offers
an option to request the reporting of sex chromosome aneuploidy (SCA). This product must not be used as thesole basis for diagnosis or other pregnancy management decisions.
The VeriSeq NIPT Solution v2 includes: the VeriSeq NIPT Workflow Manager v2 for the VeriSeq NIPT Microlab
STAR, the VeriSeq NIPT Sample Prep and the VeriSeq Onsite Server v2 with the VeriSeq NIPT Assay Software
v2. The VeriSeq NIPT Solution v2 is intended to be used with a next generation sequencer.

Summary and Explanation of the Assay
Fetal chromosome abnormalities, specifically aneuploidy, which is an abnormal number of chromosomes, are acommon cause of reproductive failure, congenital anomalies, developmental delay, and intellectual disabilities.
Aneuploidy affects approximately 1 in 300 live births, with much higher rates associated with miscarriage andstillbirth.1,2 Until recently, there have been two types of prenatal tests for these disorders: diagnostic testing or
screening. Diagnostic testing involves invasive procedures such as amniocentesis or chorionic villus sampling.
These testing methods are considered the gold standard for detection of fetal aneuploidy. However, they areassociated with a risk of pregnancy loss between 0.11% and 0.22%.3 Conventional multiple marker screens
have no risk of pregnancy loss as they are noninvasive, but they are less accurate than diagnostic tests. Theirdetection rates for trisomy 21 vary between 69–96% depending on the particular screen, maternal age, andgestational age at testing.4 Importantly, they have false positive rates of approximately 5%, which can lead toinvasive diagnostic testing for confirmation and, thus, the risk of procedure-related pregnancy loss.4
Ultrasound screens can also detect chromosome abnormalities, but they do so with even less certainty thanthese other methods.
Fetal aneuploidy for chromosomes 21, 18, 13, X and Y can be detected with a high degree of accuracy bynoninvasive prenatal testing (NIPT) using whole-genome sequencing of cell-free DNA (cfDNA) obtained frommaternal plasma at 10 weeks gestation or later. A recent meta-analysis of multiple clinical studies reported theweighted pooled detection rates and specificities for trisomy 21 and trisomy 18 in singleton pregnancies asfollows: trisomy 21 99.7% and 99.96% and trisomy 18 97.9% and 99.96%, respectively.5 One study suggeststhat use of NIPT as a primary screen across all pregnancies could result in an 89% reduction in the number ofconfirmatory invasive procedures.6

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VeriSeq NIPT Solution v2 Package Insert
Given the significant reduction of false positive rates with NIPT compared to conventional multiple marker
screening, numerous professional medical organizations have issued opinion statements supporting several
indications for the use of NIPT.
Specifically, the International Society for Prenatal Diagnosis, the American College of Obstetricians andGynecologists (ACOG) /Society for Maternal Fetal Medicine (SMFM), the American College of Medical Genetics
and Genomics (ACMG), and the European Society of Human Genetics/American Society of Human Genetics
support offering NIPT to all pregnant women.7,8,9 Pretest counseling, informed consent, and diagnostic testing
to confirm a positive cfDNA screening result are recommended.4
The VeriSeq NIPT Solution v2 is a noninvasive in vitro diagnostic (IVD) test that utilizes whole-genome
sequencing of cfDNA fragments derived from maternal peripheral whole blood samples from pregnant womenof at least 10 weeks gestation. The test offers two options for types of screening: basic and genomewide. The
basic screening provides information on the aneuploidy status of chromosomes 21, 18, 13, X, and Y only.
Genomewide screenings provide partial duplications and deletions for all autosomes and aneuploidy status for
all chromosomes. Both screening types provide the option for sex chromosome aneuploidy (SCA) reporting withor without fetal sex reporting. The reporting option for SCA can be turned off. If the reporting option for SCA isturned off, fetal sex is not reported either. For more information on the sex reporting options, refer to the
VeriSeq NIPT Solution v2 Software Guide (document # 1000000067940).

Principles of Procedure
The VeriSeq NIPT Solution v2 is an automated solution for laboratory NIPT testing which consists of automatedsample preparation and sequencing data analysis. The VeriSeq NIPT Sample Prep are specialized single use
reagents that are used in conjunction with the VeriSeq NIPT Microlab STAR to prepare 24-, 48-, or 96-sample
batches for next generation sequencing. Whole genome, paired-end sequencing data is analyzed by
specialized software, the VeriSeq NIPT Assay Software v2, and a report is generated that provides qualitative
results.
The workflow consists of the following procedures: sample collection, plasma isolation, cfDNA extraction,library preparation, library quantification, library pooling, sequencing, and analysis which are outlined in moredetail:•Sample Collection—7–10 ml of maternal peripheral whole blood is collected in a Streck cell-free DNA Blood
Collection Tube (BCT), which prevents cell lysis and genomic contamination and stabilizes whole blood.

•

Plasma Isolation—Within 5 days of collection, plasma is isolated from maternal peripheral whole bloodusing standard centrifugation techniques. The VeriSeq NIPT Microlab STAR aspirates and dispenses plasma
into a 96-well deep-well plate for subsequent processing. In the event that retesting is required, postprocessing samples may be recapped and stored at 4°C for an additional 5 days (up to a total of 10 days
after blood collection).
CAUTION
Exceeding the aforementioned storage times can negatively impact individual sample failure
rates.

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VeriSeq NIPT Solution v2 Package Insert•

cfDNA Extraction—Purification of cfDNA from plasma is achieved by adsorption onto a binding plate,washing the binding plate to remove contaminants, and eluting.

•

Library Preparation—The purified cfDNA fragments undergo an end repair process to convert 5’ and 3’
overhangs to blunt ends. Next, a deoxyadenosine nucleotide is added to the 3’ ends to create a single baseoverhang. Indexed adapters containing a single base 3’ deoxythymidine overhang are then ligated onto theprocessed cfDNA fragments. The ligated DNA is purified using solid phase reverse immobilization beads.
Each sample in a set of 24, 48, or 96 receives a unique indexed adapter. The adapters serve 2 purposes:

CAUTION
Take extreme care to avoid cross contamination of the indexes which could lead to incorrect
results.

–

Indexes allow sample identification in subsequent sequencing.

–Index adapters contain sequences that allow for library capture on the solid surface of a sequencing
flow cell for cluster generation and subsequent sequencing.

•

Quantification—The library product is quantified using a fluorescent dye with concentration determined bycomparison to a DNA standard curve.

•

Library Pooling and Sequencing—The sample libraries are pooled together into 24- or 48-sample pools inadjusted amounts to minimize variation in coverage. Each pool is then sequenced using a next-generation
sequencing system.

•

The VeriSeq NIPT Solution v2 does not include sequencing equipment and consumables.

•

Analysis—For each sample, analysis consists of the following:–

Identification of library fragments by index sequence and alignment of the paired end reads to a humanreference genome.

–

Estimation of the fetal fraction of the library by combining information from the distribution of both thelengths and genomic coordinates of the library fragments.

–

After accounting for known biases, a statistical model detects regions of the genome which are under oroverrepresented in the library in a manner consistent with an anomaly at the estimated level of fetal
fraction.

–

The NIPT report provides summary results for the selected test menu where ANOMALY DETECTED or
NO ANOMALY DETECTED is listed along with a fetal fraction estimate for samples passing QC.

–

The Supplementary Report provides quantitative metrics which characterize each detected anomaly.

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VeriSeq NIPT Solution v2 Package Insert

Limitations of the Procedure
Limitations of the Assay•

Evidence supporting sensitivity and specificity for the test covers singleton and twin pregnancies. These
instructions for use do not provide sensitivity or specificity data for triplets or higher-order pregnancies.

•

The VeriSeq NIPT Solution v2 is not intended to detect polyploidy, such as triploidy.

•

The VeriSeq NIPT Solution v2 is not intended to detect balanced chromosome rearrangements.

•

The assay requires maternal peripheral whole blood samples from pregnant women of at least 10 weeks
gestation.

•

For basic screens, the VeriSeq NIPT Solution v2 test looks for specific chromosome abnormalities. Results
reported as NO ANOMALY DETECTED do not eliminate the possibility of chromosomal abnormalities of thetested chromosomes. A negative result does not eliminate the possibility that the pregnancy has otherchromosomal abnormalities, genetic conditions, or birth defects (eg, open neural tube defect).

•

For genomewide screens, large deletions and duplications that are less than 75% of the size of thechromosome can be indicative of whole chromosome aneuploidy.

•

For genomewide screens, certain regions are excluded from analysis. A list of such blacklisted regions isavailable on the Illumina Support website. Genomic anomaly detection is only performed on non-excluded
regions.

•

Fetal sex reporting is not available in all regions due to local regulations governing gender reporting.

•Based on the literature evidence, cell-free DNA-based screening results can be confounded by certain
maternal and fetal factors. Some of these are listed below, but are not limited to the following:

–
–
–
–
–
–
–
–

Recent maternal blood transfusion
Maternal prior organ transplant / stem cell transplant
Maternal autoimmune disease
Maternal neoplasms (benign and malignant)Maternal mosaicism
Maternal copy number variations
Fetoplacental mosaicism / confined placental mosaicism
Fetal demise / vanishing twin

VeriSeq NIPT Solution v2 Reporting•

The VeriSeq NIPT Solution v2 is a screening test and should not be considered in isolation from other clinicalfindings and test results. Conclusions about the fetal condition and pregnancy management decisions
should not be based on the results of the NIPT screening alone.7•

The VeriSeq NIPT Solution v2 reports on the following:

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VeriSeq NIPT Solution v2 Package Insert––

Basic screen tests the overrepresentation of chromosomes 13, 18, and 21.
Genomewide screen tests under and overrepresentation of all autosomes, including partial deletions
and duplications of at least 7 Mb.

–

In singleton pregnancies with Yes or SCA selected as the sex reporting option, the following sex
chromosomal anomalies: XO, XXX, XXY, and XYY.

––

In singleton pregnancies with Yes selected as the sex reporting option, fetal sex is reported.
The presence of a Y chromosome in twin pregnancies.

Product Components
The VeriSeq NIPT Solution v2 (part # 20030577) consists of the following sample prep:•

VeriSeq NIPT Sample Prep (24 samples) (part # 20025895)•

VeriSeq NIPT Sample Prep (48 samples) (part # 15066801)•

VeriSeq NIPT Sample Prep (96 samples) (part # 15066802)

The VeriSeq NIPT Solution v2 (part # 20030577) consists of the following software components:•

VeriSeq NIPT Assay Software v2 (part # 20047024), preinstalled on the VeriSeq Onsite Server v2.

–

VeriSeq Onsite Server v2 (part # 20028403 or 20047000) or an existing VeriSeq Onsite Server (part #15076164 or # 20016240) that is upgraded to v2.

•

VeriSeq NIPT Workflow Manager v2, (part # 20044988), preinstalled on the VeriSeq NIPT Microlab STAR.

–

VeriSeq NIPT Microlab STAR (part # Hamilton Company Reno: 95475-01 (115 V) & 95475-02 (230 V),
Hamilton Company Bonaduz: 806288).

•

Local Run Manager VeriSeq NIPT module (part # 20044989)

Reagents
Reagents Provided
Illumina provides the following reagents: VeriSeq NIPT Sample Prep (24 samples) (part # 20025895), VeriSeq
NIPT Sample Prep (48 samples) (part # 15066801), and VeriSeq NIPT Sample Prep (96 samples) (part #15066802). The VeriSeq NIPT Sample Prep are configured for use with the VeriSeq NIPT Microlab STAR (MLSTAR)(part # 95475-01, 95475-02, or 806288), which is provided by the Hamilton Company.

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VeriSeq NIPT Solution v2 Package Insert
VeriSeq NIPT Sample Prep, Extraction Box
Table 1 VeriSeq NIPT Extraction Box (24) and (48), Part # 20025869 and 15066803
Reagent Nameon Label
Lysis Buffer

Number ofContainers in

Active Ingredients

Storage

Guanidine hydrochloride in buffered aqueous solution

15°C to

Kit
1

30°C
Wash Buffer I
Wash Buffer II

11

Guanidine hydrochloride and 2-propanol in buffered

15°C toaqueous solution

30°C

Buffered aqueous solution-containing salts

15°C to30°C

Elution Buffer

1

Buffered aqueous solution

15°C to30°C

Proteinase

1

Glycerol in buffered aqueous solution

BufferProteinase K

15°C to30°C

3Lyophilized Proteinase K

15°C to30°C

Table 2 VeriSeq NIPT Extraction Box (96), Part # 15066807
Reagent Name on
Label

Number ofContainers

Active Ingredients

Storage

Guanidine hydrochloride in buffered aqueous

15°C tosolution

30°C

Guanidine hydrochloride and 2-propanol in buffered

15°C toaqueous solution

30°C

Buffered aqueous solution-containing salts

15°C toin KitLysis Buffer
Wash Buffer I
Wash Buffer II

1
1
2

30°C
Elution Buffer

1

Buffered aqueous solution

15°C to30°C

Proteinase Buffer

1

Glycerol in buffered aqueous solution

15°C to30°C

Proteinase K

4

Lyophilized Proteinase K

15°C to30°C

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VeriSeq NIPT Solution v2 Package Insert
VeriSeq NIPT Sample Prep, Library Prep Box
Table 3 VeriSeq NIPT Library Prep Box (24) and (48), Part # 20026030 and 15066809
Reagent Name on
Label

Number ofContainers in Kit

End Repair Mix

1

A-Tailing Mix

1

Ligation Mix

1

Active Ingredients

Storage

DNA polymerase and dNTPs in buffered-25°C to -aqueous solution

15°C

DNA polymerase and dATP in buffered-25°C to -aqueous solution

15°C

DNA ligase in buffered aqueous solution-25°C to 15°C

Hybridization

1

Buffered aqueous solution

Buffer-25°C to 15°C

NIPT DNA

1

Adapter Plate

Oligonucleotides in buffered aqueous-25°C to -solution

15°C

Table 4 VeriSeq NIPT Library Prep Box (96), Part # 15066810
Reagent Name on
Label

Number ofContainers in Kit

End Repair Mix
A-Tailing Mix
Ligation Mix

12
2

Active Ingredients

Storage

DNA polymerase and dNTPs in buffered-25°C to -aqueous solution

15°C

DNA polymerase and dATP in buffered-25°C to -aqueous solution

15°C

DNA ligase in buffered aqueous solution-25°C to 15°C

Hybridization

1

Buffered aqueous solution

Buffer
NIPT DNA
Adapter Plate

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1

Oligonucleotides in buffered aqueous-25°C to -solution

15°C

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VeriSeq NIPT Solution v2 Package Insert
VeriSeq NIPT Sample Prep, Accessory Box
Table 5 VeriSeq NIPT Accessory Box, Part # 15066811
Reagent

Number ofName onContainers

Label

Active Ingredients

Storage

Propylene microplate with modified silicone membrane

2°C toin KitDNA Binding

1

Plate

8°C

Resuspension

1

Buffered aqueous solution

2°C toBuffer8°C

Sample1

Purification

Solid-phase paramagnetic beads in buffered aqueous

2°C tosolution

8°C

DNA intercalating dye in DMSO

2°C to

Beads
DNA

1

Quantification

8°C

Reagent
DNA

1

Quantification

dsDNA standard, nonspecific DNA, and sodium azide in

2°C tobuffered aqueous solution

8°C

Standard

VeriSeq NIPT Sample Prep, Workflow Tubes and Labels
Table 6 Workflow Tubes and Labels, Part # 15071543
Item Name on Label

Number of Items in Kit

StorageLabel (LBL)–Plate Barcode

915°C to 30°C

Label (LBL)–Deep-well Plate Barcode

1215°C to 30°C

Tube (TB)–Empty Pooling Tube

515°C to 30°C

Reagents Not Provided
Reagents Required, Not Provided•

Sequencing reagents and consumables required for next generation sequencing (NGS) system•

Certified DNase/RNase-free water•

Ethanol, 100% (200 proof) for molecular biology grade

NOTE

Non-molecular biology grade ethanol can potentially negatively impact performance of the assay.

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VeriSeq NIPT Solution v2 Package Insert
Reagents Optional, Not Provided•

Dulbecco's Phosphate-Buffered Saline (DPBS) for no template control (NTC)

Storage and Handling1.

Room temperature is defined as 15°C to 30°C.

2. All reagents are for one-time use only. After reagents are prepared for use, they should be used
immediately.
3. If any of the packaging or contents of the VeriSeq NIPT Solution components are damaged or compromised,please contact Illumina Customer Service.
4. Reagents are stable when stored as indicated until the specified expiration date on the kit labels. For
storage conditions, refer to the Storage column in the tables in Reagents on page 5. Do not use expired
reagents.
5. Changes in the physical appearance of the reagents provided can indicate deterioration of the materials. Ifchanges in the physical appearance occur (eg, obvious changes in reagent color or cloudiness apparent
with microbial contamination), do not use the reagents.
6. Adhere to the following best practices when handling Sample Purification Beads:•

Never freeze the beads.

•

Allow the beads to reach room temperature before use.

•

Immediately before use, vortex the beads until well-suspended and color appears homogeneous.

7. Lysis Buffer, Wash Buffer I, Wash Buffer II, Elution Buffer, and Proteinase Buffer can form visible precipitatesor crystals. Before use, vortex vigorously, and then visually inspect to make sure that no precipitates arepresent.
8. Never freeze whole blood after collection.
9. Sequence libraries as soon as possible after pooling. Pooled libraries are stable for up to seven days at 25°C to -15°C. No additional denaturation is required if stored for this period of time at these conditions.

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VeriSeq NIPT Solution v2 Package Insert

Equipment and Materials
Equipment and Materials Required, Not Provided
Equipment Required, Not Provided
Equipment

Supplier

A next generation sequencing (NGS) system

Instrument supplier or Illumina, part # 20005715

with the following capabilities:• 2 x 36 bp paired-end sequencing• Compatible with VeriSeq NIPT Sample Prep
dual index adapters• Automatic production of BCL files• Two channel chemistry• 400 million paired-end reads per run• Compatible with VeriSeq NIPT Assay
Software v2 or a NextSeq 550Dx
Sequencing System.
Freezer, -25°C to -15°C

General lab supplier

Microcentrifuge

General lab supplier

Pipette aid

General lab supplier

Refrigerator, 2°C to 8°C

General lab supplier

20 µl single-channel pipettes

General lab supplier

200 µl single-channel pipettes

General lab supplier1000 µl single-channel pipettes

General lab supplierVortexer

General lab supplier

Centrifuge and rotor assembly for blood collection tubes

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VeriSeq NIPT Solution v2 Package Insert
Equipment

Supplier

Equivalent:• Refrigerated centrifuge capable of 1600 × g

General lab supplierwith no-brake option• Swinging bucket rotor with buckets• Bucket inserts, 24, 48, or 96 tube capacity,
76 mm minimum depth• Insert adapters to support 16 x 100 mm
blood collection tubes
Recommended:• Allegra X12R Series Centrifuge, 1600 g• Allegra Centrifuge GH-3.8 Rotor withbuckets• Allegra Centrifuge Bucket Covers, set oftwo• Allegra Centrifuge Adapter Assembly, 16mm, set of four

Beckman Coulter, item #392304 (120 V or 230 V)
Beckman Coulter, item #369704
Beckman Coulter, item #392805
Beckman Coulter, item #359150

Centrifuge and rotor assembly for microplates
Equivalent:• Centrifuge capable of 5600 × g• Swinging plate rotor with 96-well plate
carriers, 76.5 mm minimum depth.
• Multifuge X4 Pro-MD 120V TX-1000BT• Sorvall Legend XTR Centrifuge• HIGHPlate 6000 Microplate Rotor• Rotor high plate 6000
Support base for microplates• Recommended:• MicroAmp 96-Well Support Base
• 96-Well PCR Plate Carrier

General lab supplier

Thermo Scientific VWR catalog number 76326-254
Thermo Fisher Scientific, catalog # 75004521 (120 V) or
catalog # 75004520 (230 V)
Thermo Fisher Scientific, catalog # 75003606
Thermo Scientific VWR catalog number 97040-244

Thermo Fisher Scientific, catalog # 4379590
Thermo Fisher Scientific, catalog # AB-0563/1000

One of the following microplate readers, or
equivalent, (fluorometer) with SoftMax Pro
v6.2.2 or higher:• Gemini XPS• SpectraMax M2, M3, M4, and M5.
• Purple insert is included with microplate

Molecular Devices, part # XPS
Molecular Devices, part # M2, M3, M4, and M5

reader for use in workflow.

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VeriSeq NIPT Solution v2 Package Insert
Equipment

Supplier

SpectraMax High-Speed USB, Serial Adapter

Molecular Devices, part # 9000-0938

Thermal cycler with the following

General lab supplierspecifications:• Heated lid• 4°C to 98°C temperature range• ±2°C temperature accuracy• 2°C per second minimum ramp rate
• Compatible with Twin.tec PCR Plate 96well, full skirt
VeriSeq NIPT Microlab STAR

Hamilton, part # 95475-01 (115 V), part # 95475-02 (230 V),
or part #806288 (for Hamilton Company Bonaduz)

VeriSeq Onsite Server v2 or an upgraded

Illumina, part # 20028403 or 20047000 (v2) or #15076164

VeriSeq Onsite Server

or # 20016240 (upgraded)

If using a NextSeq 550Dx Sequencing System:• NextSeq 550Dx High Output Reagent Kit

Illumina, part # 20028870

v2.5, 75 cycles

Optional Equipment, Not Provided
Equipment

Supplier

Pluggo Decapper System

LGP Consulting, part # 4600 4450

SpectraMax SpectraTest FL1 fluorescence

Molecular Devices, part # 0200-5060

validation plate
Tube Revolver/Rotator, 15 ml tubes, 40 rpm,

Thermo Scientific, catalog # 88881001 (US) or catalog

100–240 V# 88881002 (EU)Materials Required, Not Provided
Consumable

Supplier

1000 μl Conductive Non-Sterile Filter Tips

Hamilton, part # 235905

300 μl Conductive Non-Sterile Filter Tips

Hamilton, part # 235903

50 μl Conductive Non-Sterile Filter Tips

Hamilton, part # 235948

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VeriSeq NIPT Solution v2 Package Insert
Consumable

SupplierDeep-well reservoir with the following specifications:• SLAS 1–2004 microplate format with 96 pyramidal or

General lab supplierconical bottom wells and a 240 ml minimum capacity.
• Polypropylene with preference for low DNA binding for all

Compatible reservoirs:• Corning Axygen, product # RES-

sample contact surfaces.
• Internal dimensions (liquid level) are compatible with

SW96-HP-SI• Agilent, product # 201246-100

automated aspiration and dispensing steps of VeriSeq
NIPT Microlab STAR.
• Height dimensions are compatible with automatedmovements of VeriSeq NIPT Microlab STAR.
Reagent tub with the following specifications:• Tub that fits securely, but not forced, into the carrier ofthe VeriSeq NIPT Microlab STAR with tapered bottom anda 20 ml minimum capacity.
• Polypropylene that is free from RNase/DNase.
• Internal reservoir dimensions (liquid level) generate liquid

General lab supplierCompatible tubs:• Roche, product # 03004058001

levels using assay reagent volumes that are arecompatible with automated aspirating and dispensing
steps of VeriSeq NIPT Microlab STAR.
• Height dimensions are compatible with automatedmovements of VeriSeq NIPT Microlab STAR.
Deep-well plates with the following specifications:• SLAS 1–2004, 3–2004, and 4–2004 microplate format
with 96 pyramidal or conical bottom wells and a 2 ml
minimum well capacity.
• Translucent polypropylene, with preference for low DNA
binding material for all sample contact surfaces.
• Well dimensions generate liquid level that are compatible

General lab supplierCompatible plates:• Eppendorf, part # 0030505301• Eppendorf, part # 30502302• USA Scientific, part # 1896-2000

with automated aspiration and dispensing steps of
VeriSeq NIPT Microlab STAR.
• Plate skirt that permits placement of plate barcodes torequire position with secure, flate surface adhesion.
• Torque-resistant frame able to sustain a minimum of 5600
x g.
• Plate height dimensions are compatible with automatedmovements of VeriSeq NIPT Microlab STAR

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VeriSeq NIPT Solution v2 Package Insert
Consumable

Supplier

384-well plate with the following specifications:• Microplate with 384 wells, optimized for low-volumes,

General lab supplierwith a 50 µl minimum well capacity.
• Black opaque polystyrene with light-blocking and low

Compatible plates:• Corning, product # 3820

DNA binding for all sample contact surfaces.
• Well dimensions generate liquid levels that are compatible
with automated aspiration and dispensing steps of
VeriSeq NIPT Microlab STAR.
• Plate height dimensions are compatible with automatedmovements of VeriSeq NIPT Microlab STAR.
• Plate skirt that permits placement of plate barcodes torequired position with secure, flat surface adhesion.
96-well plate with the following specifications:• Microplate with a torque-resistant frame able to sustain aminimum of 5600 x g and 96 translucent wells withtapered bottoms, raised rims, and a 150 µl minimum wellcapacity.
• Polypropylene that is free from RNase/DNase with low
DNA binding for all sample contact surfaces.
• Well dimensions generate liquid levels that are compatible
with automated aspirating and dispensing steps ofGeneral lab supplierCompatible plates:• Eppendorf, part # 0030129512• Eppendorf, part # 30129580• Eppendorf, part # 30129598• Eppendorf, part # 30129660• Eppendorf, part # 30129679• Bio-Rad, part # HSP9601

VeriSeq NIPT Microlab STAR.
• Plate height dimensions are compatible with automatedmovements of VeriSeq NIPT Microlab STAR.
• Plate skirt that permits placement of plate barcodes torequired position with secure, flat surface adhesion.
• Compatible with thermal cyclers for denaturing.
One of the following seals:• Microseal 'F' Foil
• Foil seals

Bio-Rad, catalog # MSF1001
Beckman Coulter, item # 538619

Cell-Free DNA BCT CE

Streck, catalog # 218997

Push Caps

Sarstedt, order # 65.802

2 ml Screw-cap tubes

General lab supplier

20 µl filter tips for 20 µl pipettor

General lab supplier

200 µl filter tips for 200 µl pipettor

General lab supplier1000 µl filter tips for 1000 µl pipettor

General lab supplier

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VeriSeq NIPT Solution v2 Package Insert
Consumable

Supplier

Equivalent:• An alcohol-based rapid disinfectant spray• A solution of disinfecting detergent
Recommended:• De-ionized water and 70% ethanol

General lab supplier

Optional Materials, Not Provided
Consumable

Supplier

Dulbecco's Phosphate-Buffered Saline (DPBS) for no

General lab suppliertemplate control (NTC)
Tube, screw cap, 10 ml (for control samples only)

Sarstedt, order # 60.551

Tube, screw cap, 50 ml

General lab supplier

25 ml serological pipettes

General lab supplier

10 ml serological pipettes

General lab supplierSpecimen Collection, Transport, and Storage
CAUTION
Handle all specimens as if they are potentially infectious agents.

•

Whole blood specimens of 7–10 ml must be collected in Streck Cell-Free DNA BCT.

•Transportation of whole blood must comply with all applicable governing regulations for the transport ofetiologic agents. Expedited shipping/transportation methods are recommended.

•

During transport, store at temperatures between 4°C and 30°C. After samples are received, store at 2°C to
8°C until ready to proceed. The time between blood collection and initial plasma isolation should not exceed
5 days.

•In the event that retesting is required, post-processing samples may be recapped and stored at 4°C for an
additional 5 days (up to a total of 10 days after blood collection).

CAUTION
Exceeding the aforementioned storage times can negatively impact individual sample failure
rates.

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Warnings and Precautions•

This assay contains Proteinase K. Personal injury can occur through inhalation, ingestion, skin contact, andeye contact. Use in a well-ventilated area, wear protective clothing, avoid breathing dust, and dispose of
any containers and unused contents in accordance with applicable governmental safety standards.

•

This assay contains guanidinium chloride. Personal injury can occur through inhalation, ingestion, skincontact, and eye contact. Use in a well-ventilated area, wear protective clothing, and dispose of any
containers and unused contents in accordance with applicable local governmental safety standards.

•

This assay contains 2-propanol, a flammable chemical. Keep away from heat and open flames. Personal
injury can occur through inhalation, ingestion, skin contact, and eye contact. Use in a well-ventilated area,
wear protective clothing, and dispose of any containers and unused contents in accordance with applicable
local governmental safety standards.

•

This assay contains dimethyl sulfoxide, a corrosive and combustible liquid. Personal injury can occur
through inhalation, ingestion, skin contact, and eye contact. Use in a well-ventilated area, wear protective
clothing, and dispose of any containers and unused contents in accordance with applicable local
governmental safety standards.

•

To prevent the formation of harmful gases, do not dispose of cfDNA extraction waste (contains guanidinehydrochloride) with waste containing bleach (sodium hypochlorite).

•

Handle all specimens as if they contain potentially infectious agents.

•

Use routine laboratory precautions. Do not pipette by mouth. Do not eat, drink, or smoke in designated work
areas. Wear disposable gloves and laboratory coats when handling specimens and assay reagents. Wash
hands thoroughly after handling specimens and assay reagents.

•

Do not use any assay components beyond their stated expiration date on the assay box label. Do notinterchange assay components from different assay lots. Assay lots are identified on the assay box label.
Store the assay components at the specified temperature.

•

To prevent sample or reagent degradation, make sure that all sodium hypochlorite vapors from cleaning
have fully dissipated before starting the protocol.

•

Failure to follow the procedures as outlined can result in erroneous results or significant reduction in sample
quality.

•

Immediately report any serious incidents related to this product to Illumina and the Competent Authoritiesof the member states in which the user and the patient are established.

•

For environmental, health, and safety information, refer to the safety data sheets (SDS) at
support.illumina.com/sds.html.

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Procedural Notes
Avoiding Contamination•

Use fresh tips and fresh consumable labware.

•

Use aerosol-resistant tips to reduce the risk of carry‐over and sample‐to‐sample cross‐contamination.

•

Due to the potential for contamination, take extreme care to make sure that well contents remain fully in thewell. Do not splash contents. Centrifuge following any vortexing step.

•

Follow applicable governing regulations for proper laboratory practice and hygiene when handling bloodand blood derivatives.

•

Do not use aerosol bleach sprays when performing library preparation. Trace bleach contamination can leadto assay failure.
• When unsealing any plates, take care to place the plate on a firm, flat surface, gripping the plate firmly.
Slowly remove the seal ensuring that the seal does not make contact with exposed wells. Take care to nottouch exposed wells or disturb contents. Well to well cross-contamination can produce incorrect results.

VeriSeq NIPT Microlab STAR Deck Cleaning•

Before use, inspect the deck for cleanliness. At least once a week, conduct the weekly maintenance andfollow these cleaning instructions.

•Remove all unloadable carriers and clean with an alcohol-based rapid disinfectant spray De-ionized waterand 70% ethanol and leave them to dry. If they are heavily soiled, soak them afterward in a solution ofdisinfecting detergent, rinse with the alcohol-based disinfectant, and leave to dry.

•Open the front cover and wipe the deck with a cloth saturated with De-ionized water and 70% ethanol. The
slide blocks in particular must be checked for cleanliness.

•

Remove the Basic Vacuum System (BVS) manifold and clean manifold, gasket, and interior compartments of
BVS with cloth. Avoid cleaning gasket with ethanol as this can brittle the material.

•

Empty tip waste for the CORE 96-head and the independent channel.

•

Remove the independent channel tip eject plate of the tip waste station and clean it: spray De-ionized waterand 70% ethanol directly onto the surface and wipe. Pull a new plastic bag over the frame and re-attach it.
Put the clean tip eject plate back in place.

•Spray De-ionized water and 70% ethanol directly onto the surface of the CORE 96-head waste box andchute and wipe it clean.

–

If buildup is difficult to remove from tip wastes, wipe with a cloth wet with DNase/RNase-free water until
the buildup is removed. Dispose of the cloth appropriately. Proceed to sterilize with the alcohol-based
disinfectant.

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Wet a lint-free cloth or cotton swab with 70% ethanol. Swab the laser scanner window of the barcode
reader. Using the same cloth or swab, clean each well of the CPAC plate adapter. If using a cloth, press thecloth into each well of the adapter using the back of a pen to assure the interior of the well is properly
cleaned.

•

Clean the independent channels:–

On the independent channels, clean the tip eject sleeve (outer part of the pipetting channels) with a lintfree cloth soaked in De-ionized water and 70% ethanol. (See the Hamilton Microlab STAR Reference
Guide #15070074.)–

Clean the stop disk and the O-rings of the pipetting head (outer part of the pipetting channels) with alint-free cloth soaked in De-ionized water and 70% ethanol.

•

Clean the CORE 96-head:–

Using the same lint-free cloth soaked in De-ionized water and 70% ethanol, clean the casing of the 96head and the bottom of the stop disks.

–

Using the same cloth, or a torn strip of cloth soaked in De-ionized water and 70% ethanol, ‘floss’ thecloth around the sides of the pipette channels of the 96-head in order to clean the o-rings. Repeat thisprocedure for every pipette channel on the 96-head.

•

Spray the front and side cover with De-ionized water and 70% ethanol, and wipe dry.

•

Clean the Autoload protecting ribbon with a cloth soaked in De-ionized water and 70% ethanol, and wipe
without exerting pressure.

•

When the deck and components are completely dry, replace the carriers.

NOTE

Improper cleaning and maintenance of the ML STAR can result in cross-contamination and poor
assay performance.

Quality Control
Control material with known performance characteristics may be evaluated to detect differences in processing
and technical procedures in the laboratory.
Running a control sample or no template control reduces the total number of unknown maternal samples thatcan be processed with each sample prep.
Do not exceed two NTC samples per batch of 24 or 48 samples or four NTC samples per batch of 96 samples.

Instructions for Use
Tips and Techniques
Unless a safe stopping point is specified in the protocol, proceed immediately to the next step.

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Barcoding Plates•

Barcodes for full-skirt plates start with PL.

•

Barcodes for deep-well plates start with DW.

•

Apply barcodes to full-skirt plates and deep-well plates on the side next to column 12.

•

Load plates with the barcode facing right to enable automated scanning.

Sealing and Unsealing the Plate•

Take extreme care to avoid cross contamination - there should be no visible liquid on underside of the seal.

––•

Take care to not touch exposed wells.

Always seal the 96-well plate before the following steps in the protocol:––•

Ensure that the exposed underside of the seal does not make contact with exposed wells.

Centrifuge steps
Thermal cycling steps

To seal the plate, apply the foil seal to the plate and then seal. Ensure that pressure is applied across theentire plate and the seal is tight across each individual well.

•

Before unsealing the plate, perform the following:––

Briefly centrifuge the 96-well plate at 1000 × g for 20 seconds.
Place the plate on a flat surface, and then remove the seal slowly.

VeriSeq NIPT Microlab STAR•

Before use, perform and document the required maintenance according to the manufacturer instructions.

•Observe the ML STAR during the automated steps. Monitor the VeriSeq NIPT Workflow Manager v2
software interface for prompts and operator instructions.

•

Keep the front cover in place during operation.

•

Keep the deck clear of all items during operation.

•

If presented with the option button Exclude during an error handling event, refrain from choosing this option
under any circumstances. If the method cannot precede past the error handling event or have limited error
handling options, abort the run.

•

During plate vacuum steps, if prompted by the VeriSeq NIPT Workflow Manager v2, manually assist forming
the seal between the plate and the vacuum manifold.

•

Allow the system to dispose of tips from the adapter automatically. Do not manually remove tips unlessprompted by the software.

•

Remove spent reagents and used consumables as prompted by the Workflow Manager.

•

Empty vacuum waste carboys daily. The first carboy should never exceed ½ full. Overflow of vacuum waste
can damage the vacuum pump and reduce the applied vacuum of the system.

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For 24, 48, and 96 sample batches, load a full rack of the individually counted 8-channel tips prior tostarting method.

Process Samples
Procedure
1.

Complete the following steps for each aliquot:
a. Centrifuge barcoded samples at 1600 × g for 10 minutes at 4°C with the brake off.
b. Remove the sample tubes when the centrifuge comes to a complete stop.
Begin plasma isolation within 15 minutes after centrifugation. If more than 15 minutes elapse, centrifuge
again.

2. Inspect each tube for sample suitability by verifying the following requirements:•

Sample volume is as expected.

•

Clear separation between the red blood cell and plasma layers of the samples are visible after
centrifugation.

•

Plasma level is at least 1.5 ml above the buffy coat.

•

Sample is not heavily hemolyzed (ie, plasma is not deep red in appearance).

•

Sample is not lipemic (ie, plasma is not cloudy white or milky opaque in appearance).

•

Sample does not have clotting.

CAUTION
Samples that have been improperly stored or handled can become unsuitable. If unsuitablesamples are processed through the workflow, they can clog the binding plate during extractions
and cause samples to overflow into adjacent wells.
3. Uncap the tubes and load them into the tube carriers. Load all samples and any plasma controls for thebatch.

CAUTION
During an error-handling event, if presented with the Exclude option, do not select it. If themethod cannot proceed past the error-handling event and you have limited error-handling
options, abort the run.

Isolate Plasma
Preparation
1.

Label 1 deep-well plate Intermediate Plasma and apply a barcode.

2. Label 1 deep-well plate Final Plasma and apply a barcode.
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3. For 24, 48, and 96 sample batches, load a full rack of the individually counted 8-channel tips prior tostarting the method.

CAUTION
Make sure to use the correct plate type for the Intermediate Plasma and Final Plasma plates. Useof a deep-well reservoir instead of a deep-well plate leads to sample amalgamation and can
produce incorrect results.

Procedure
1.

Open the AppLauncher, and then select VeriSeq NIPT Method.

2. Enter a unique Batch ID and user name, and then select OK.
Batch ID can contain ≤ 26 characters. You can use numbers, letters, underscores (_), or dashes (-). For
example: 2025-10-16_Batch3.
The Batch ID is not case-sensitive. Case-sensitive Batch IDs are not considered unique.
Batch names must be unique and must not differ only in capitalization. For example, batch names Batch01
and batch01 are not unique. This same rule applies for Sample ID naming.
3. Select New Batch.
4. After initiation, select OK to begin plasma isolation.
5. Perform one of the following steps:•

To load an existing sample sheet, select the sample sheet associated with the batch, and then selectOK.

•

To proceed without selecting a sample sheet, select No Sample Sheet.

For information on creating a sample sheet, refer to VeriSeq NIPT Solution v2 Software Guide (document #1000000067940).

NOTE

Sample type, singleton, or twins, must be accurately recorded for each sample to ensure proper
data analysis. If you choose No Sample Sheet, make sure you set default sample values in the
Workflow Manager Service Tools. Refer to VeriSeq NIPT Solution v2 Software Guide (document #1000000067940) for more information.

6. Select the batch size, and then select OK.
7. Select the number of no template controls (NTCs), and then select OK.
NTC slots are always the last slots selected. For example, with two NTCs in a 24-sample run, positions 23
and 24 are NTCs.

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8. Confirm that all barcodes are affixed, and then load the samples, tips, and plates (barcode-facing right)
onto the carrier.
9. Select OK after each load prompt.
SampleBatch
Size24, 48, 96

Carrier
Type
Tip

Tube

Site

Track

Item

7–12

1000 µl tips

51000 µl tips (96 batch only)

4, 5

Prepared blood sample tubes 1–24 (for all batch

1–24

15

Positionsizes)
Tube

16

Prepared blood sample tubes 25–48 (48 and 96

25–48

batch size only)
Tube

17

Prepared blood sample tubes 49–72 (96 batch size

49–72

only)
Tube

18

Prepared blood sample tubes 73–96 (96 batch size

73–96

only)
Multiflex

19–24

Empty deep-well plate, Final Plasma – barcoded

4

Multiflex

19–24

Empty deep-well plate, Intermediate Plasma –5barcoded
Reagent

47[Optional] Dulbecco's Phosphate-Buffered Saline

5(DPBS) - used for no template control (NTC)
10. Make sure that the carriers, labware, and reagents are loaded correctly.
11. On the Pre-Spin Deck Verification screen, select OK.
12. Observe the ML STAR perform the automated steps.
13. When prompted by the Workflow Manager, make sure the ML STAR loading deck is free of any obstructions
to allow the ML STAR to unload the carriers.
14. Select Unload to unload the deck.
15. Remove the Intermediate Plasma deep-well plate as follows.
a. Inspect the plate for consistent volumes in each well (no pipette errors). The expected volume is 1000
µl.
b. Record any inconsistencies when the Plasma Isolation procedure is complete.
c. Seal the plate, load with balance, and centrifuge at 5600 × g for 10 minutes with the brake off or on thelowest setting.
16. Select Yes to proceed to final Plasma Preparation.

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17. Remove the plate seal and reload the plate onto the carrier.
Sample Batch Size
24, 48, 96

Carrier
Type
Multiflex

Track

Item

19–24

Intermediate Plasma deep-well plate

Site
Position5

18. Select the Intermediate Plasma plate has been spun checkbox, and then select OK.
19. Observe the ML STAR perform the automated steps.
20. When prompted by the Workflow Manager, make sure that the ML STAR loading deck is free of any
obstructions to allow the ML STAR to unload the carriers.
21. Select Unload to unload the deck.
22. When prompted by the Workflow Manager, empty the carriers and deck.
23. Remove the Final Plasma deep-well plate.
24. Inspect the plate for the following errors:•

inconsistent volumes in each well. Expected volume is 900 µl.

•

Visible cell pellets.

•

Excessive hemolysis.

If you observe abnormal visible cell pellets or excessive hemolysis, invalidate the affected sample at theend of the Plasma Isolation method or use Batch Manager. For more information about Batch Manager, refer
to VeriSeq NIPT Solution v2 Software Guide (document # 1000000067940).
25. When prompted by the Workflow Manager, select OK.
26. Enter comments about affected wells, and then select OK.
27. Perform one of the following steps.

•

To proceed to cfDNA Extraction, select Yes.

•

To stop, select Exit.

SAFE STOPPING POINT
If you are stopping, seal the Final Plasma plate and store at 2°C to 8°C for up to 7 days.

Extract cfDNA
Preparation
1.

Visually examine the Extraction and Accessory Boxes to confirm that the kit is not expired.

2. Prepare the following reagents. Label the reservoir tubs and deep-well reservoirs with the name of thereagents.

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Reagent

Storage

Instructions

Final Plasma deep-well

2°C toIf previously stored, let stand for 30 minutes to bring to room

plate

8°C

temperature.
Centrifuge at 1000 x g for 20 seconds.
Unseal the Final Plasma deep-well plate before use.

3. Slowly add 3.75 ml Proteinase Buffer to each reagent vial of Proteinase K.

•

Prepare 3 vials for 24 and 48 samples.

•

Prepare 4 vials for 96 samples.

4. Cap the Proteinase K vials and vortex until resuspended.

CAUTION
Do not contaminate the rubber stopper. Getting other substances on the rubber stopper cancontaminate future samples.
5. Pool the prepared Proteinase K from all vials into a reagent tub and label it as Proteinase K.
6. Add 100 ml 100% EtOH to each reagent bottle of Wash Buffer II.

•

Prepare 1 bottle for 24 and 48 samples.

•

Prepare 2 bottles for 96 samples.

7. Invert the Wash Buffer II bottles to mix.
8. Mark the checkboxes on the Wash Buffer II bottles.
9. Label 1 new full-skirt plate Intermediate and apply a plate barcode.
10. Label 1 new full-skirt plate cfDNA Elution and apply a plate barcode.
11. Label 1 new deep-well plate Extraction Intermediate and apply a deep-well plate barcode.
12. Apply a plate barcode to the DNA Binding plate.
13. Apply a foil seal to unused wells for 24 and 48 sample bathes.
14. Prepare a 70% EtOH cleaning solution (70% EtOH, 30% DNase/RNAse-free water) for cleaning the vacuum
system.
15. Prepare the vacuum system as follows.
a. Remove the vacuum manifold and clean with 70% EtOH.
Avoid cleaning the gasket with EtOH as this can brittle the material.
b. Empty the vacuum waste.
c. Make sure that the ML STAR vacuum system is on.

Procedure1.

Select OK to start cfDNA Extraction.

2. If the VeriSeq NIPT Method is not open:

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a. Open the AppLauncher, and then select VeriSeq NIPT Method.
b. Enter the Batch ID and user name, and then select OK.
3. Load tips onto the tip carriers as follows, and then select OK.

CAUTION
Before starting method for 24, 48, and 96 sample batches, add full rack of 8-channel tips.
Sample Batch Size
24

48

96

Carrier
Type
Tip

Tip

Tip

Track

Item

Site Position

1–6

1000 µl tips

1

7–12

300 µl tips

1

1–6

1000 µl tips

1, 2

7–12

300 µl tips

1

1–6

1000 µl tips

1, 2, 3, 4

7–12

300 µl tips

1

Track

Item

49–54

1000 µl tips

1

300 µl tips

2

50 µl tips

3

4. Load counted tips onto the tip carriers as follows.
Sample Batch Size
24, 48, 96

CarrierType
Tip

Site Position

5. Enter the location of the first and last tips for each tip rack, and then select OK.

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6. Scan the Extraction Box barcodes.
7. Enter the user name or reagent preparer initials, and then select OK.
8. Scan the Accessory Box barcodes.
9. Enter the user name or reagent preparer initials, and then select OK.
10. Confirm that barcodes are affixed.
11. Unseal the Final Plasma deep-well plate if necessary.
12. Load plates (barcode-facing right) onto the plate carrier as follows, and then select OK.
Sample Batch

Carrier

Size

Type

24, 48, 96

Multiflex

Site

Track

Item

19–24

New full-skirt plate, Intermediate, barcoded1

New full-skirt plate, cfDNA Elution, barcoded2

New deep-well plate, Extraction Intermediate,

4

Positionbarcoded
Final Plasma deep-well plate, barcoded

5

13. Confirm that the DNA Binding plate is barcoded, then select OK.
14. For partial plate batches, apply a trimmed plate seal over the unused wells (columns 4–12 for 24 sample
batches and columns 7–12 for 48 sample batches).
15. Load the DNA Binding plate onto the vacuum manifold with the barcode-facing right.
16. Before placing the binding plate on the BVS manifold, visually inspect the wells for any possible
obstructions.
This can impede the flow of reagents whilst under vacuum.
17. If using 24 or 48 sample batches, cover unused wells and seal with foil seal. Select the Are DNA Binding
Plate Columns Sealed? checkbox, and then select OK.
18. Load reagent tubs onto the reagent carrier as follows, and then select OK.
Sample Batch Size
24, 4896

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CarrierType
Reagent

Reagent

Track
47

47

Item

Site Position

16 ml Elution Buffer1

11 ml Proteinase K

2

16 ml Elution Buffer1

15 ml Proteinase K

2

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VeriSeq NIPT Solution v2 Package Insert
19. Transfer the specified reagents into the deep-well reservoirs, and then load the reservoirs onto the deepwell carriers as follows.
20. Select OK.
Carrier

Sample Batch Size
24, 48

96

Track

Item

39–44

125 ml Wash Buffer II

1

125 ml Wash Buffer I

2

60 ml 100% EtOH

3

100 ml Lysis Buffer

4

60 ml DNase/RNase-free water5

200 ml Wash Buffer II

1

125 ml Wash Buffer I

2

100 ml 100% EtOH

3

100 ml Lysis Buffer

4

100 ml DNase/RNase-free water

5

Type
Deep-well

Deep-well

39–44

Site Position

21. Wait for the automated reagent volume check to complete.
22. Confirm that the vacuum waste is empty (half full recommended), and then select OK.
23. Confirm the placement of all carriers, labware, and reagents, and then select OK in the Extraction Deck
Verification screen.
24. Observe the ML STAR during the automated steps.
You must manually invalidate sample overflows not detected by the system prior to thecontamination of nearby wells.
25. After the final vacuum step, remove the DNA Binding plate and clean the bottom surface with 70% EtOH.
26. Seal any uncovered wells on the DNA Binding plate, and then place the DNA Binding plate on the empty
Final Plasma deep-well plate.
27. Centrifuge the DNA Binding plate/Final Plasma plate assembly at 5600 × g for 10 minutes with the brake on.
28. Select OK.
29. During DNA Binding plate centrifugation, complete the vacuum cleaning:a. Remove vacuum manifold, and then select OK.
b. Wait for the automated waste disposal to complete.
c. Clean the vacuum manifold and inside the vacuum system with 70% EtOH, and then replace the vacuum
manifold.
d. Select the Manifold is on Vacuum checkbox to initiate the elution plate transfer on vacuum manifold,
and then select OK.

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30. After centrifugation, unseal the wells containing samples on the DNA Binding plate.
31. Place the DNA Binding plate on top of the cfDNA Elution plate that is on the vacuum manifold.
32. Load the DNA Binding plate with the barcode to the right, and then select OK.
33. Observe the ML STAR during the automated steps.
34. After the incubation step, select the Plates are assembled as indicated checkbox. Confirm that the DNA
Binding/cfDNA Elution plate assembly is on a support base (if required by centrifuge).
35. Seal the uncovered wells on the DNA Binding plate.
36. Centrifuge at 5600 × g for 2 minutes with the brake on, and then select OK.
37. Visually inspect the cfDNA Elution plate for consistent volumes in each well.
The expected volume is approximately 55 µl.
38. Seal and retain the cfDNA Elution plate for library preparation.
39. When prompted by the Workflow Manager, make sure that the ML STAR loading deck is free of any
obstructions to allow the ML STAR to unload the carriers.
40. Select Unload to unload the deck.
41. Unload all carriers and clean the ML STAR deck, and then select OK.
42. Enter comments about affected wells, and then select OK.
43. Perform one of the following steps:•

To continue to Prepare Libraries, select Yes.

•

To stop, select Exit.

SAFE STOPPING POINT
If you are stopping, seal the cfDNA Elution plate and store at -25°C to -15°C for up to 7 days.

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Prepare Libraries
Preparation
1.

Visually examine the Library Prep and Accessory boxes to confirm that the kits are not expired.

2. Prepare the following reagents. Label the reservoir tubs and deep-well reservoirs with the reagent names.
Reagent

Storage

Instructions

A-Tailing Mix-25°C to -15°C

Thaw at room temperature. Vortex to mix, andthen centrifuge briefly.

cfDNA Elution Plate-25°C to -15°C

If previously stored, confirm that the plate was
not stored more than 7 days and thaw at room
temperature. Vortex at 1500 rpm for 1 minute.
Centrifuge at 1000 × g for 20 seconds.

End Repair Mix-25°C to -15°C

Thaw at room temperature. Vortex to mix.

Hybridization Buffer-25°C to -15°C

Thaw at room temperature. Vortex to mix. Returnto storage after use.

Ligation Mix-25°C to -15°C

Thaw at room temperature. Vortex to mix, andthen centrifuge briefly.

NIPT DNA Adapter-25°C to -15°C

Plate

Thaw at room temperature. Vortex to mix.
Centrifuge at 1000 × g for 20 seconds.

Resuspension Buffer

2°C to 8°C

Vortex to mix. Return to storage after use.

Sample Purification

2°C to 8°C

Let stand for 30 minutes to bring to room

Beads

temperature.
Vortex vigorously before each use. Mix by
vortexing or inversion until all beads are insuspension and the mixture is homogenous.

CAUTION
When unsealing the NIPT DNA adapter plate take extreme care to avoid well to well aerosol crosscontamination which can produce incorrect results.
3. If the cfDNA Elution plate was stored frozen, prepare it as follows.
a. Thaw at room temperature.
b. Vortex at 1500 rpm for 1 minute.
c. Centrifuge at 1000 × g for 20 seconds.
4. Label one new full-skirt plate Libraries and apply a plate barcode.
5. Prepare 80% EtOH from absolute EtOH. Combine 40 ml 100% EtOH and 10 ml DNase/RNase-free water.
Invert to mix.
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6. Make sure that the ML STAR thermal control is on.

Dilute Enzymes
1.

Combine A-Tailing Mix and Resuspension Buffer in a screw cap tube. Vortex to mix, and then centrifuge
briefly.
Sample Batch Size

A-Tailing Mix (µl)

Resuspension Buffer (µl)

24, 48

900

1200

96

1800

2400

2. Combine Ligation Mix and Resuspension Buffer in a screw cap tube. Vortex to mix, and then centrifuge
briefly.
Sample Batch Size

Ligation Mix (µl)

Resuspension Buffer (µl)

24, 48

230

1713

96

440

3278

Procedure1.

Select OK to start Library Preparation. If the VeriSeq NIPT Method Method is not already open:
a. Open the AppLauncher and select VeriSeq NIPT Method.
b. Enter the Batch ID and user name, and then select OK.

2. Confirm that the following consumables are prepared, as indicated in the Reagent Preparation screen:•

A-Tailing Mix, Ligation Mix, and 80% EtOH•

Sample Purification Beads, End Repair Mix, and NIPT DNA Adapter Plate

3. Select the checkboxes, and then select OK.
4. Scan the Library Prep Box barcodes.
5. Enter the user name or reagent preparer initials, and then select OK.
6. Scan the Accessory Box barcodes.
7. Enter the user name or reagent preparer initials, and then select OK.

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8. Load tips onto the tip carriers as follows, and then select OK for each carrier.
Carrier

Sample Batch Size

Type

24

Tip

48

Tip

96

Tip

Track

Item

Site Position

1–6

50 µl tips

1

7–12

300 µl tips

1, 2

1–6

50 µl tips

1, 2

7–12

300 µl tips

1, 2, 3, 4

1–6

50 µl tips

1, 2, 3, 4

7–12

300 µl tips

1, 2, 3, 4, 5

9. If you stopped the protocol after the cfDNA Extraction procedure, load counted tips onto the tip carriers as
follows.
Carrier

Sample Batch SizeType24, 48, 96

Tip

Track

Item

Site Position

49–54

1000 µl tips

1

300 µl tips

2

50 µl tips

3

10. Enter the location of the first tip for each tip rack, and then select OK.
11. Confirm that barcodes are affixed, and load plates (barcode facing right) onto the plate carrier as follows,
and then select OK.
Sample Batch

Carrier

Size

Type

24, 48, 96

Multiflex

Site

Track

Item

19–24

cfDNA Elution plate, barcoded

1

NIPT DNA Adapter plate, barcoded2

New 96 well full-skirt plate, libraries,

3

Positionbarcoded
New 96 well full-skirt plates

4, 5

12. Load the deep-well carrier as follows, and then select OK.
Sample Batch Size
24, 48, 96

CarrierType
DeepwellDocument # 1000000078751 v08
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Track

Item

39–44

50 ml 80% EtOH in a deep-well reservoir
New 96 well full-skirt plates

Site
Position12, 3, 4, 5

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13. Load reagent tubs onto the reagent carrier as follows, and then select OK.
Carrier

Sample Batch Size
24, 48, 96

Type
Reagent

Track
47

Item

Site Position

2.5 ml End Repair Mix

1

Prepared A-Tailing Mix (total volume)

2

Prepared Ligation Mix (total volume)3

10 ml Sample Purification Beads

4

12 ml Hybridization Buffer

5

14. Save the remainder of the 12 ml hybridization buffer (HT1) in the container for pooling.
15. Make sure that the carriers, labware, and reagents are loaded as indicated, and then select OK in the Library
Deck Verification screen.
16. Wait for the automated reagent volume check to complete.
17. Observe the ML STAR during the automated steps.
18. When prompted by the Workflow Manager, make sure that the ML STAR loading deck is free of any
obstructions to allow the ML STAR to unload the carriers.
19. Select Unload to unload the deck.
20. Inspect the Libraries plate for consistent volumes in each well.

CAUTIONIf well volumes are inconsistent, samples can produce incorrect results.
21. If storing, seal and retain the Libraries plate.
22. Unload the carriers, clean the deck, and then select OK.
23. Enter comments about affected wells, and then select OK.
24. Perform one of the following steps:•

To continue to Quantify Libraries, select Yes.

•

To stop, select Exit.

SAFE STOPPING POINT
If you are stopping, seal the Libraries plate prior to storage. The Libraries plate is stable for up to 7 days fromdate of preparation at -25°C to -15°C.

Quantify Libraries
Preparation1.

Prepare the following reagents:

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Reagent

Storage

Instructions

DNA Quantification

2°C to 8°C

Protect from light. Thaw at room temperature for 30–150

Reagent

minutes.
(Removing reagent at start of the Prepare Libraries procedure isrecommended.)
Vortex to mix, and then centrifuge briefly.

DNA Quantification

2°C to 8°C

Vortex to mix, and then centrifuge briefly.

2°C to 8°C

Vortex to mix.

Standard
Resuspension Buffer

2. If the Libraries plate was stored frozen, prepare it as follows.
a. Confirm that the plate was not stored for more than 7 days and thaw at room temperature.
b. Vortex to mix
c. Centrifuge at 1000 × g for 1 minute.
3. Turn on the fluorometer 10 minutes before use.
4. Apply a plate barcode to a new 384-well plate.
5. Apply a plate barcode to a new full-skirt plate.

Procedure1.

Select OK to start quantification.

2. If the VeriSeq NIPT Method is not already open:
a. Open the AppLauncher, and select VeriSeq NIPT Method.
b. Enter the Batch ID and user name, and then select OK.
3. Scan the Accessory Box barcodes.
4. Enter the user name or reagent preparer initials, and then select OK.
5. Load tips onto the tip carrier as follows, and then select OK.
Sample Batch Size
24, 4896

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Carrier
Type
Tip

Tip

Track

Item

1–6

300 µl tip rack1

50 µl tip rack2

300 µl tip rack1

50 µl tip rack

2, 3

1–6

Site Position

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6. Confirm that barcodes are affixed.
7. If needed, unseal the Libraries plate.
8. Load plates (barcode-facing right) onto the Multiflex carrier as follows, and then select OK.
Carrier

Sample Batch Size
24, 48, 96

TypeMultiflex

Track

Item

Site Position

19–24

New full-skirt plates, barcoded1

New 384-well plate, barcoded2

Libraries plate, barcoded3

New 96-well full-skirt plates

4, 5

9. Load reagent tubes without caps into the tube carrier as follows, and then select OK.
Sample Batch Size
24, 48, 96

Carrier

Track

Type
Tube

46

Item

Site Position

DNA Quantification Standard

1

DNA Quantification Reagent

2

10. Load reagent tubs onto the reagent carrier as follows, and then select OK.
Sample Batch Size
24, 48, 96

Carrier

Track

Type
Reagent

47

Item

Site Position

New reagent tub (empty)

1

16 ml Resuspension Buffer2

11. If you stopped the protocol after the Library Preparation procedure, load counted tips onto the tip carriers
as follows.
Sample Batch Size
24, 48, 96

Carrier
Type
Tip

Track

Item

Site Position

49–54

1000 µl tips

1

300 µl tips

2

50 µl tips

3

12. Enter the location of the first and last tips for each tip rack, and then select OK.
13. Make sure that the carriers, labware, and reagents are loaded as indicated, and then select OK in the Quant
Deck Verification screen.
14. Wait for the automated reagent volume check to complete.
15. Observe the ML STAR during the automated steps.
16. When prompted by the Workflow Manager, make sure that the ML STAR loading deck is free of any
obstructions to allow the ML STAR to unload the carriers.
17. Select Unload to unload the deck.
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18. Unload the Libraries plate.
a. Inspect the plate for consistent volumes in each well.
b. Seal the Libraries plate and store at room temperature until the fluorometric data analysis is complete.
19. Unload the remaining 96-well plates and check for consistent volumes in each well.
Gross errors in volume can indicate an issue with pipetting steps.
20. Unload the 384-well plate and check for liquid in the appropriate wells.
21. Seal the plate with a foil seal.
22. Centrifuge at 1000 × g for 20 seconds.
23. Incubate at room temperature for 10 minutes, protected from light.
24. Unload all carriers.
25. Clean the ML STAR deck, and then select OK.

CAUTION
Do not discard quantification reagents until data is obtained. You require the reagents if you needto perform requantification.
26. After incubation, remove the foil seal and load the 384-well plate onto the microplate reader. Make sure to
use the purple adapter plate (Part Number: 0310-4336) provided by Molecular Devices or equivalent if
applicable by instrument used.

•

Make sure that A1 is in the top-left corner when loading.

27. Double select the VeriSeq NIPT template to open it in SoftMax Pro.
28. Select New Experiment in the Home tab.
29. Select Read.
30. Export the data as XML as follows.
a. Right select Plate, and then select Rename.
b. Scan the barcode of the Quantification plate, and then select OK.
c. In the upper-left corner of the screen, select the plate icon, and then select Export from the menu.
d. Select the Expt name checkbox, set the plate date option to raw, set the output format to XML, and thenselect OK.
e. Set the output file path and name, and then select Save.
The Hamilton computer must be able to access the file location. Do not use spaces in the file name or filepath.

Analysis1.

On the ML STAR, in the Scanner Information screen, enter the fluorometer ID.

2. Enter comments about the fluorometer run, and then select OK.
3. Navigate to the *.xml quantification file that contains the fluorometric data, and then select OK.

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4. Review the standards curve and sample concentration analysis results, and then select OK.
5. If you must rescan the plate, select Rescan.
Samples are time and light sensitive. When necessary, perform the rescan immediately.
6. Enter comments about affected wells, and then select OK.
7. Assess the results and proceed as follows.

•

If the results pass specification, proceed to Pool Libraries on page 36. For specifications, refer to thequantitation QC metrics and boundaries table in the VeriSeq NIPT Solution v2 Software Guide(document # 1000000067940).

•

If the results fail specification, the system aborts the method. Repeat the quantification procedures
beginning with Preparation on page 32.

8. Perform one of the following steps:•

To proceed to Pool Libraries on page 36, select Yes.

•

To stop, select Exit.

SAFE STOPPING POINT
If you are stopping, seal the Libraries plate prior to storage. The Libraries plate is stable for up to 7 days
cumulative storage at -25°C to -15°C.

Pool Libraries
Preparation1.

Prepare the following reagents:
Reagent

Storage

Instructions

Hybridization-25°C toThaw at room temperature. Vortex to mix. Return to storage after

Buffer

-15°C

use.

2. If the Libraries plate was stored frozen, prepare it as follows.
a. Confirm that the plate was not stored for more than 7 days and thaw at room temperature.
b. Vortex at 1500 rpm for 1 minute.
c. Centrifuge at 1000 × g for 20 seconds.
d. Pipette to mix.
3. Label an empty pooling tube Pool A. For 96 samples, label a second empty pooling tube Pool B.

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4. Save the following denature program on the thermal cycler with a heated lid.
a. Choose the preheated lid option and set to 102°C.
b. Set the reaction volume to 50 μl.
c. Set the ramp rate to maximum (≥ 2°C per second).
d. Incubate at 96°C for 10 minutes, and then 4°C for 5 seconds.
e. Hold at 4°C.

Procedure
1.

Place the Libraries plate on the preprogrammed thermal cycler and run the denature program.
Do not denature Libraries plate before the quantification has passed QC metrics, as you may want toperform requantification.

2. Centrifuge the Libraries plate at 1000 × g for 20 seconds.
3. Select OK to start pool libraries.
4. If the VeriSeq NIPT Method is not open:
a. Open the AppLauncher, and select VeriSeq NIPT Method.
b. Enter the Batch ID and user name, and then select OK.
5. Select the pool concentration, and then select OK.
The target cluster density is 220–260 K/mm².

NOTE

Pooling concentrations and/or pooling volumes may need to be increased for 24 sample batches
to maintain similar cluster densities obtained with 48/96 sample batches.

6. If prompted by the Workflow Manager, perform one of the following steps:•

To load a sample sheet, select the sample sheet associated with the batch, and then select Load.

•

To use system default values for remaining sample types, sex reporting, or screen type, select Use
Default for each setting.
For information about creating a sample sheet, refer to the VeriSeq NIPT Solution v2 Software Guide(document # 1000000067940).

7. Select Start to begin timer for denaturing plate.
8. Load tips onto the tip carriers as follows.
Sample Batch Size
24, 48, 96

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Carrier
Type
Tip

Track

Item

7–12

50 µl filter tips

Site Position1

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9. Load the Denatured Library plate (barcode facing right) onto the Multiflex carrier as follows, and then selectOK.
Sample Batch Size
24, 48, 96

Carrier
Type
Multiflex

Track

Item

Site Position

19–24

Denatured Library plate (barcoded)

1

10. Load pooling tubes onto the tube carrier as follows, and then select OK.
Carrier

Sample Batch Size

Type

Track

Item

Site Position

24, 48Tube

46

New 2 ml tube, Pool A

1

96

Tube

46

New 2 ml tube, Pool A

1

New 2 ml tube, Pool B

2

11. Load reagent tubs onto the reagent carrier as follows, and then select OK.
Sample Batch Size
24, 48, 96

CarrierType
Reagent

Track
47

Item

Site Position

3 ml Hybridization Buffer

1

12. Load tips onto the tip carriers as follows.
Sample Batch Size
24, 48, 96

Carrier
Type
Tip

Track

Item

Site Position

49–54

1000 µl filter tips

1

300 µl filter tips

2

50 µl filter tips

3

13. Enter the location of the first and last tips for each tip rack, and then select OK.
14. Make sure that the carriers, labware, and reagents are loaded as indicated.
15. In the Pooling Deck Verification screen, select OK.
16. Observe the ML STAR during the automated steps.
17. Enter comments about the affected wells, and then select OK.
18. When prompted by the Workflow Manager, make sure that the ML STAR loading deck is free of any
obstructions to allow the ML STAR to unload the carriers.
19. Select Unload to unload the deck.
20. Unload the tube carrier.
21. Cap each pooling tube, vortex, and then centrifuge briefly.
22. Select OK.
23. Sequence libraries as soon as possible after pooling. Seal the Libraries plate and store at -25°C to -15°C for
up to 7 days to allow repooling.
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SAFE STOPPING POINT
If you are stopping, cap the pooling tubes and store at -25°C to -15°C for up to 7 days.

Prepare Pooled Libraries for Sequencing
Preparation1.

Prepare the following reagents:
Reagent

Storage

Instructions

Pool tubes-25°C to -15°C

If previously stored, thaw at room temperature.
Vortex briefly.
Centrifuge briefly.

2. Prepare the next-generation sequencing system by completing the following fields in the Local Run
Manager VeriSeq NIPT Module module:a. Run Nameb. [Optional] Run Descriptionc. Pool Barcode

CAUTION
The Pool Barcode entered in the LRM module must match the Pool Barcode entered in the
Workflow Manager. Incorrect run configurations are rejected by the analysis software and require
resequencing.
For more information on using the Local Run Manager VeriSeq NIPT Module, refer to the VeriSeq NIPT
Solution v2 Software Guide (document # 1000000067940).

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Procedure
1.

Combine the following volumes to the reagent cartridge, and then pipette to mix.

•

Hybridization Buffer (900 µl)•

450 µl Pool A (450 µl)

2. Proceed with sequencing using a next generation sequencing system, following the system guide.
reference guide for your next-generation sequencing instrument. For a NextSeq 550Dx, refer to the
NextSeq 550Dx Instrument Reference Guide (document # 1000000009513) or NextSeq 550Dx Instrument
Package Insert (document # 1000000043133).
3. Confirm correct run configuration when prompted.
4. If necessary, repeat this procedure for Pool B.

•

To achieve target cluster density range, the library plate can be repooled using a different poolingconcentration on the Hamilton. Repooling invalidates the original pool.

•

Alternatively, the ratio of pool to HT1 (450 µl + 900 µl) can be modified to achieve target cluster density
range.

Next Generation Sequencing
The VeriSeq NIPT Solution v2 can be used with a next-generation sequencing system with the following
specifications:•capable of 2x36 paired-end reads.

•

compatible with index adapters in the VeriSeq NIPT Sample Prep.

•

two channel chemistry.

•

automatic production of .BCL files (raw data from sequencing instrument).

•

400 million paired-end reads per run.

•compatible with VeriSeq NIPT Assay Software v2.

The NextSeq 550Dx is compatible with the VeriSeq NIPT Solution v2.

Sequence Data Analysis
After sequencing is complete, sequencing data is automatically sent to the VeriSeq NIPT Assay Software v2 foranalysis and report generation. The report includes classifications for each sample in the batch as well as an
assessment of all run QC metrics. The analysis process from sequencing completion to final results takes
approximately 4 hours for a 48-sample batch. For detailed information on data analysis and the output file, referto the VeriSeq NIPT Solution v2 Software Guide (document # 1000000067940).

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Interpretation of Results
The VeriSeq NIPT Solution v2 algorithm employs a sophisticated statistical model that combines several
different types of information from the collection of paired-end sequenced library fragments. This model isused to detect regions of the genome that are under or overrepresented in the library of each sample.
Importantly, this model accounts for whether the degree of under or overrepresentation is quantitatively
consistent with an aneuploid event in the fetal genome at the level of fetal fraction estimated for the library.
For all chromosomes, paired-end sequencing data are aligned with the reference genome (HG19). Unique
nonduplicated aligned reads are aggregated into 100 kb bins. The corresponding bin counts are adjusted for GC
bias and according to previously established region-specific genomic coverage. Using such normalized bin
counts, statistical scores are derived for each autosome by comparing the coverage regions that can beaffected by aneuploidy with the rest of the autosomes. A log likelihood ratio (LLR) is computed for each sample
by taking into account these coverage-based scores and the estimated fetal fraction. The LLR is the probabilityof a sample being affected given the observed coverage and fetal fraction versus the probability of a sample
being unaffected given the same observed coverage. The calculation of this ratio also takes into account theestimated uncertainty in fetal fraction. For subsequent calculations, the natural logarithm of the ratio is used.
The Assay Software assesses the LLR for each target chromosome and each sample to provide a determinationof aneuploidy.
During batch creation, you must define the type of a sample (singleton or twin), the screening type (basic or
genomewide), and the sex chromosome reporting (Yes, No, and SCA) desired for each sample. Together, these
options determine the information reported for each sample.
For all sample types, the screening type determines which autosomal anomalies are reported. For the basic
screening type, only whole chromosome trisomy events involving chromosomes 13, 18 and 21 are reported. For
the genomewide screening type, whole or partial chromosome deletion or duplication of any autosomal
chromosome are reported. The length of the smallest reportable partial chromosome deletion or duplication is 7
Mb.
For singleton samples, you can disable sex chromosome reporting. You can also configure to report sex
chromosome aneuploidies either with or without reporting the sex of euploid samples.
For twin samples, if Yes is selected for sex chromosome reporting, the result is limited to reporting the presenceor absence of a Y chromosome in the library. Sex chromosome aneuploidy cannot be reported for twin samples.
A result of ANOMALY DETECTED indicates the sample screens positive for one or more anomalies consistent
with the selected screen type and sex chromosome reporting option. When an anomaly is detected, the reportprovides a description of the anomaly in cytogenetic notation.
The VeriSeq NIPT Assay Software v2 uses statistics generated during sequencing to provide a fetal fraction
estimation (FFE) for each sample. The FFE is the estimated fetal cfDNA component that is recovered by theassay and reported as a rounded percentage for each sample. The average standard deviation of this estimate
across all samples is 1.3%. The FFE is not to be used in isolation to exclude samples when reporting results.

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To make chromosomal representation calls, the VeriSeq NIPT Assay Software v2 uses the individualized Fetal
Aneuploidy Confidence Test (iFACT), a dynamic threshold metric that indicates whether the system has
generated sufficient sequencing coverage, given the fetal fraction estimate for each sample. Negative calls arereported only if the sample meets the iFACT threshold. If a sample fails to achieve this threshold, the QC
assessment displays FAILED iFACT and the system does not generate a result.
In addition to iFACT, the VeriSeq NIPT Assay Software v2 assesses several other QC metrics during analysis.
The additional metrics include assessments of coverage uniformity on reference genomic regions and the
distribution of cfDNA fragment lengths. The QC assessment displays either a QC flag or a QC failure for any
metrics outside of the acceptable range. In the case of QC failure, the system does not generate a result for the
sample. If a sample fails QC, the sample can be reprocessed provided sufficient plasma volume is in the bloodcollection tube.
The VeriSeq NIPT Solution v2 generates data for use in a final report. It does not generate a final report for thepatient. Customers are responsible for the design and contents of the final report to be delivered to the point ofcare physician. Illumina is not responsible for the accuracy of wording in the final report for customers.

CAUTION
Check the fetal fraction estimates of all samples. If fetal fraction estimates are similar for all samples
within a run, sample amalgamation may have occurred and impacted results. Contact Illumina
Technical Support for help troubleshooting.

Performance Characteristics
The following data outlined in the clinical performance and analytical performance sections were generated byusing the protocols and materials outlined in the Instructions for Use starting with plasma. All sequencing data
for this section were generated on a NextSeq 500/550 sequencing system or a NextSeq 550Dx sequencing
system with the following configurations:
NextSeq 500/550

NextSeq 550Dx

NextSeq Control Software 4.0

NextSeq Operating Software 1.3

Reagent Kit

NextSeq 500/550 High Output v2.5 (75

NextSeq 550Dx High Output v2.5 (75Versioncycle) Reagent Kit

cycle) Reagent Kit

Sequencing

2x36 paired-end sequencing run in high2x36 paired-end sequencing run in highMethodoutput mode

output mode

On-instrument
SoftwareClinical Study
The clinical accuracy of the VeriSeq NIPT Solution v2 was demonstrated by evaluating plasma samples frompregnant women with singleton and twin pregnancies. Samples were obtained from deidentified banked plasma
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VeriSeq NIPT Solution v2 Package Insert
considered for study inclusion. These samples underwent previous prenatal screening for fetal chromosome
aneuploidies and partial deletions and duplications of 7 Mb or greater. All samples from affected pregnancies
and a subset of consecutive samples from unaffected pregnancies were eligible for testing if clinical outcomes
were available and sample criteria were met. A total of 2,335 samples were in the testing analysis set. From thisset, 2,328 samples were from singleton pregnancies and seven samples were from twin pregnancies.
Of these samples, 28 (1.2%, 28/2335) samples failed assay QC on first pass during the analysis of thecompleted sequencing data:•

27 iFACT failures (one XO, 26 unaffected)•

One failure for data outside of expected range

Demographics and Pregnancy Characteristics
Maternal age, gestational age, and trimester of pregnancy are summarized in Table 7 for the samples in the
genomewide screen, including known mosaic samples. The majority (98%) of testing samples represent firsttrimester pregnancy.
The demographics were assessed between the basic and genomewide cohorts and showed no statisticaldifference. Demographics and pregnancy characteristics were similar whether known mosaics were included or
excluded.

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Table 7 Demographics and Pregnancy Characteristics
Summary Statistic
Number of samples

Genomewide(including known mosaics)
2307*

Maternal age – years
Mean

35.08

Standard Deviation

4.04

Median34.95

25th percentile, 75th percentile

32.31, 37.79

Minimum, maximum

20.22, 53.02

Gestational age at blood draw - weeks
Mean

10.93

Standard Deviation

1.20

Median10.57

25th percentile, 75th percentile

10.29, 11.14

Minimum, maximum

10.00, 27.86

Trimester of pregnancy – n (%)< First (<14 weeks)

2,252 (98%)

Second54 (2%)

Third (≥ 27 weeks)

1 (0%)* Final samples presented contained 7 twins.

Clinical Performance
Results as called by the VeriSeq NIPT Solution v2 were compared to the clinical reference standard outcomes.
All study samples had clinical reference standard outcomes (clinical truth) related to fetal chromosomal
aneuploidy status and partial deletions and duplications of 7 Mb or greater. The clinical reference standard
outcome for samples included in this study depended on results of chromosome analysis or a newborn physicalexam with an NGS-based NIPT negative screen. Trained study staff performed classification of clinicalreference standard data in accordance with the Medical Coding document from the sponsor.

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Chromosome analysis methods included karyotyping, fluorescence in situ hybridization (FISH), or comparative
genomic hybridization chromosome microarray (CMA). Chromosome analysis was performed on neonatal or
infant peripheral blood or saliva, products of conception (POC) samples, amniocytes, chorionic villi, placental
tissues, or postnatal umbilical cord blood.
Mosaicism is defined as the presence of two or more cell lines of different chromosomal composition in an
individual. The cell lines originate from the same zygote. The type and level of mosaicism varies and isdependent on the timing of mosaic events during embryogenesis and fetal development. Different types ofmosaicism appear in prenatal diagnoses depending on the distribution of abnormal versus normal cell lines overcytotrophoblast, mesenchyme, or the fetus.10 Although mosaicism can be seen with any chromosomal anomaly,
the prevalence of mosaicism in rare trisomies is higher than in the trisomies of chromosomes 21, 18, and 13 (T21,
T18, and T13).11 In the performance evaluation, mosaic cases were included in genomewide analysis, as the
purpose of this screen type for this assay is to detect rare autosomal aneuploidies (RAAs).

Basic Screen Performance
For the basic screen, anomalies include T21, T18, and T13. A total of 2,243 singleton and twin samples were
included in the analysis. All seven twin pregnancies were correctly detected as T21 and are not reported in thefollowing table.
Table 8 Sensitivity and Specificity of the VeriSeq NIPT Solution v2 for Detecting Trisomies 21, 18, & 13 in a
Basic Screen for Singleton Pregnancies (Excluding Known Mosaics)
T21

T18

T13

Sensitivity> 99.9% (130/130)> 99.9% (41/41)> 99.9% (26/26)

2-sided 95% CI

97.1%, 100%

91.4%, 100%

87.1%, 100%

Specificity

99.90% (1982/1984)99.90% (1995/1997)

99.90% (2000/2002)

2-sided 95% CI

99.63%, 99.97%

99.64%, 99.97%

99.64%, 99.97%

The assay performance in the basic screen as shown in Table 8 is calculated excluding a subset of 64 samples
affected by RAAs, autosomal partial deletions or duplications, or known mosaicism. These 64 samples included
eight T21 and three T18 mosaics. Five of these 11 samples were identified as affected with the anomaly
detected by the VeriSeq NIPT Assay Software v2.

Genomewide Screen Performance
For the genomewide screen, any anomaly includes trisomies, monosomies, and partial deletions or duplicationsof 7 Mb or greater. Samples for the genomewide screen contained 36 samples with known mosaicism. A total of
2,307 singleton and twin samples were tested. All seven twin pregnancies were correctly detected as having achromosome 21 anomaly and are not reported in the following tables.

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Genomewide Screen Performance for Any Anomaly
Table 9 Sensitivity and Specificity of the VeriSeq NIPT Solution v2 for Detecting Any Anomaly in the
Genomewide Screen (Including Known Mosaics)
Sensitivity

Specificity

Estimate % (n/N)

95.5% (318/333)

99.34% (1954/1967)

2-sided 95% CI

92.7%, 97.3%

98.87%, 99.61%

Genomewide Screen Performance for Rare Autosomal Aneuploidy
Table 10 Sensitivity and Specificity of the VeriSeq NIPT Solution v2 for Rare Autosomal Aneuploidy (RAA) in the
Genomewide Screen (Including Known Mosaics)
Sensitivity

Specificity

Estimate % (n/N)

96.4% (27/28)

99.80% (2001/2005)

2-sided 95% CI

82.3%, 99.4%

99.49%, 99.92%

Genomewide Screen Performance for Partial Deletions and Duplications
Table 11 Sensitivity and Specificity of the VeriSeq NIPT Solution v2 for Partial Deletions and Duplications of 7
Mb or More in the Genomewide Screen (Including Known Mosaics)
Sensitivity

Specificity

Estimate % (n/N)

74.1% (20/27)

99.80% (2000/2004)

2-sided 95% CI

55.3%, 86.8%

99.49%, 99.92%

Differences in Performance Between the Basic and Genomewide Screens
The scoring methodology for common trisomies and sex chromosome aneuploidies is the same for both thebasic and genomewide screens. The basic screen only applies the algorithm to T21, T18, and T13. However, thegenomewide screen expands upon this methodology to assess for all trisomies and RAAs and partial
duplications and deletions.
There are two differences between the performance reporting described between basic and genomewide
screens. First, for the genomewide screen, samples with known mosaicism both for common trisomies and for
RAAs and partial deletions and duplications were included for performance metrics. Second, the genomewide
screen can preferentially report the detection of a partial duplication or deletion over a full trisomy. The
presence of a full trisomy in addition to a partial duplication or deletion can be seen by referencing the LLR
score provided in the supplementary report.

Inclusion of Mosaics in Genomewide Screen

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Mosaicism is listed as a limitation of this assay. When mosaicism is present, the fetal signal of an anomaly is
reduced and therefore may be more challenging to detect without compromising the overall specificity of theassay. However, because mosaicism is more relevant for expanded content, samples with mosaicism were
included in the genomewide screen.
Of the 64 samples included in the genomewide screen but not the basic screen, 36 samples were identified ashaving mosaicism by the clinical reference standard. Of these 36 samples, 23 calls matched the clinicalreference standard.

Partial Deletion or Duplication versus Whole Chromosome Aneuploidy Detection
The VeriSeq NIPT Solution v2 features menu options for both a basic screen and a genomewide screen. In thebasic screen, an ANOMALY DETECTED result is only reported when a full aneuploidy is detected on
chromosomes 21, 18, or 13 and if all quality control metrics are met. In the genomewide screen, the system
detects aneuploidy across all autosomes and partial deletion and duplication events of at least 7 Mb.
While using the genomewide screen, in cases in which both a whole chromosome event as well as a CNV event
within the same chromosome exceeds the LLR threshold the system gives reporting precedence to a partial
deletion or duplication event over the whole chromosome call if the size of the partial deletion or duplication
covers approximately 75% or less of the chromosome on which the event is detected. If the partial deletion andduplication region detected is larger than 75% of the size of the chromosome, the event is reported as a full
trisomy or monosomy of the entire chromosome if simultaneously the LLR threshold for the whole chromosome
is also exceeded. Due to this, substantially large deletions and duplications that are less than or equal to 75% of
the size of the chromosome can be indicative of a whole chromosome aneuploidy.
In all samples, the LLR score for the whole chromosome classification is available in the supplementary report.
The LLR score should be reviewed with respect to the specified cutoff in 95% Detection Probabilities for
Average Regions by Size for the VeriSeq NIPT Solution v2 on page 57 prior to interpreting the result. For
example, a CNV call where the Chromosome-level LLR scores exceeding the cutoff provide further support for
an interpretation consistent with an aneuploidy of the whole chromosome, refer to Table 12 as an example.
In the clinical study, there were two singleton pregnancy samples with substantially large duplications (one on
chromosome 21 and one on chromosome 18) that were less than 75% of the relative size of the chromosome(refer to Table 12). Both events were reported as a partial duplications rather than a full trisomy for thatchromosome. The LLR scores for these events were above the cutoff consistent with an affected outcome for a
full trisomy. For either a partial duplication or a full trisomy call, the follow-up management for a positive NIPT
call is offering the patient confirmation testing via prenatal diagnosis.
Table 12 Examples of Large Duplication Events Identified in the Genomewide Screen
Clinical Truth
SampleTrisomy 21

1singleton

SampleTrisomy 18

2singleton

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Size of Anomaly

% of

LLR

Output(Mb)

Chromosome

ScoresPartial duplication on 21

22.50

48.9

19.43

Partial duplication on 18

47.00

60.2

12.99

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VeriSeq NIPT Solution v2 Package Insert
Refer to the VeriSeq NIPT Solution v2 Software Guide (document # 1000000067940) for additional information
on Quality Control metrics used to report aneuploidy results.

Sex Chromosomes
The VeriSeq NIPT Solution v2 sex chromosome results were compared to the clinical reference standard
outcome and are summarized in the following table. The percent concordance was calculated for each sex
chromosome within each clinical reference standard outcome. Percent concordance was calculated as thenumber of samples in which the VeriSeq NIPT Solution v2 sex chromosome call matched the clinical reference
standard classification, divided by the total number of samples with the same clinical reference standard
classification.
Table 13 Percent Concordance for Fetal Sex Classification*
Phenotype
Fetal Sex Classificationfrom theCytogenetic Results

Newborn
Physical Exam

Detected

Karyotyp

Femal

e

e

XX

Male

XX

XY

XO

XXX

XXY

XYY

Other**

Missing

997

0

21

0

2

0

0

0

0

0

XY

0

966

0

15

0

0

0

0

0

1

XO

0

0

0

0

19

0

0

1

0

0

XXX

0

0

0

0

0

17

0

0

1

0

XXY

0

0

0

0

0

0

23

0

1

0

XYY

0

0

0

0

0

0

0

11

0

0

Total

997

966

21

15

21

17

23

12

2

1

Percent

100

100

100

100

90.5

100

100

91.7

AnomalyNot
Detected
AnomalyNot
Detected
Anomaly
Detected
Anomaly
Detected
Anomaly
Detected
Anomaly
Detected

Not

Not

Concordan

Applicabl

Applicabltee* Five twin pregnancies were correctly classified as presence of Y. Two pregnancies were correctly classified as nopresence of Y.
** Other cytogenetic results were XXXXX and XXYY.
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Positive Predictive Value and Negative Predictive Value of the VeriSeq NIPT
Solution v2
Positive predictive value (PPV) and negative predictive value (NPV) of the test provide information regarding
the ability of the test to inform clinical decisions based on test sensitivity, specificity, and pretest probability
that a fetus is trisomy affected (prevalence). Because PPV and NPV depend on prevalence and the prevalence
for these aneuploidies can vary across different subject populations, PPV and NPV were calculated for a rangeof plausible prevalence values based on the sensitivity and specificity values observed in the basic screen(without known mosaics) of the clinical accuracy study. Table 17 is based on the genomewide screen (withknown mosaics).
Table 14 Trisomy 21 Prevalence, PPV, and NPV in Basic Screen (Excluding Known Mosaics)
Prevalence (%)

PPV (%)NPV (%)

0.05

33.17

> 99.99

0.10

49.82

> 99.99

0.20

66.53

> 99.99

0.50

83.29

> 99.99

1.00

90.93

> 99.99

1.50

93.79

> 99.99

2.00

95.29

> 99.99

Table 15 Trisomy 18 Prevalence, PPV, and NPV in Basic Screen (Excluding Known Mosaics)
Prevalence (%)

PPV (%)NPV (%)

0.03

23.06

> 99.99

0.05

33.31

> 99.99

0.10

49.99

> 99.99

0.20

66.68

> 99.99

0.30

75.03

> 99.99

0.40

80.04

> 99.99

0.50

83.38

> 99.99

Table 16 Trisomy 13 Prevalence, PPV, and NPV in Basic Screen (Excluding Known Mosaics)
Prevalence (%)

PPV (%)NPV (%)

0.01

9.10

> 99.99

0.02

16.68

> 99.99

0.05

33.37

> 99.99

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Prevalence (%)

PPV (%)NPV (%)

0.10

50.05

> 99.99

0.20

66.73

> 99.99

Table 17 Any Anomaly Prevalence, PPV, and NPV in Genomewide Screen (Including Known Mosaics)
Prevalence (%)

PPV (%)NPV (%)

0.01

1.42

> 99.99

0.02

2.81

> 99.99

0.05

6.74

> 99.99

0.10

12.64

> 99.99

0.20

22.45

99.99

0.50

42.07

99.98

1.00

59.34

99.95

1.50

68.75

99.93

2.00

74.68

99.91

Fetal Fraction Distribution
The distribution of VeriSeq NIPT Solution v2 Fetal Fraction (FF) estimates from the genomewide screen withmosaics are shown by Clinical Reference Standard outcome category in Figure 1.

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Figure 1 Fetal Fraction Distribution

FF estimates ranged from 2% to 34% overall with a median of 9% and interquartile (IQ) range of 6% to 12%. The
median FF estimate for common trisomies and events detected by the genomewide screen is 8% and for SCAs
is 9%. The range in FF estimates was consistent for all the outcomes. There is no apparent shift in the
distribution of FF among common trisomies, SCAs, events detected by the genomewide screen, or all samples
in the genomewide analysis.

Performance in Twin Pregnancies
Estimating Trisomy 13, 18, and 21 and Chromosome Y Performance in Twin
Pregnancies
Due to the low prevalence of trisomy 21, 18, and 13 in twin pregnancies, only a small number of affected twinsamples were available for the clinical study. To estimate the performance of the VeriSeq NIPT Solution v2 intwin pregnancies, in silico models based on observations from clinical samples were used to simulate
populations of twin pregnancies. This simulation was consistent with the intended use population. The
distribution of fetal fraction was determined from approximately 4,500 twin samples and compared to the
distribution from approximately 120,000 singleton samples. The distribution of fetal fraction conditional onaneuploidy status was determined from singleton putative calls (1,044 trisomy 21, 307 trisomy 18, and 192
trisomy 13). Combining the two distributions allowed for inferences of aneuploidy detection in twins. Sets ofDocument # 1000000078751 v08
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VeriSeq NIPT Solution v2 Package Insert
dizygotic and monozygotic twins were simulated, and a weighted average representing their prevalence in theintended use population was taken (2 dizygotic: 1 monozygotic) to estimate sensitivity. For specificity, sets ofunaffected twins were simulated.
The fraction of each simulated sample affected by the trisomy (ie, the affected fraction) was calculateddifferently for each sample category:•

For monozygotic twins, the affected fraction of each sample was set to 1.0 because, in this situation, thetrisomy affects both twins.

•

For dizygotic twins, it was assumed that only one twin was affected (to have both dizygotic twins beaffected is extremely rare). Affected fraction values were simulated using the known distribution of fetal
fraction ratios as determined from sex discordant clinical twin samples. A conservative approach was taken
whereby it was assumed that the affected twin always had the lowest fetal fraction of the two twins. A
correction factor was applied for fetal fractions being on average lower in trisomy 13 and 18 pregnancies.

•

For unaffected twins, the affected fraction of each sample was set to zero.

For twins affected by either trisomy 18 or 13, the fetal fraction corresponding to the affected fraction of thesample was reduced. The reduction was proportional to the average reduction in fetal fraction observed inclinical data in trisomy 18 or 13 singletons versus euploid singletons.
Both the overall fetal fraction and the affected fraction of each simulated sample were then used to calculate ananeuploidy score using the standard VeriSeq NIPT Solution v2 algorithm. Sensitivity was calculated bydetermining how often the aneuploidy scores for the simulated affected twins were above the corresponding
aneuploidy cutoff. Correspondingly, specificity was calculated by determining how often the aneuploidy scoresfor the simulated unaffected twins were below the corresponding aneuploidy cutoff (Table 18). 95% confidence
intervals were estimated based on the number of real clinical twin samples in the original data set, which wereclassified as either affected or unaffected by the relevant trisomy.
To estimate chromosome Y sensitivity in twin samples, sets of XY/XY and XX/XY twins were simulated. A
weighted average representing their prevalence in the intended use population was taken (1 XY/XY: 1 XX/XY).
To estimate chromosome Y specificity in twins, a set of XX/XX twins was simulated. The overall fetal fraction
values were simulated according to the known distribution of fetal fraction in clinical twin samples.
For XY/XY and XX/XY twins, corresponding chromosome Y scores were estimated using the known relationshipbetween fetal fraction and chromosome Y scores in clinical singleton samples classified as male. For XX/XY
twins only, affected (ie, male) fetal fraction values were simulated using the known distribution of fetal fraction
ratios observed between twins from the same pregnancy, as determined from sex discordant clinical twinsamples. A conservative approach was taken whereby the affected fraction was selected such that itcorresponded to the smaller of the two twins. For each simulated XX/XY sample, the chromosome Y score wasmultiplied by the affected fraction.
For XX/XX twins, chromosome Y scores were sampled from those scores observed in clinical singleton samples
classified as female. The chromosome Y score and the overall fetal fraction were then used to classify eachsimulated sample as chromosome Y present or chromosome Y absent using the standard VeriSeq NIPT Solution
v2 algorithm.

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VeriSeq NIPT Solution v2 Package Insert
Sensitivity was calculated by determining how often the simulated XY/XY or XX/XY twins were correctlyclassified as chromosome Y present. Specificity was calculated by determining how often the simulated XX/XX
twins were correctly classified as chromosome Y absent. 95% confidence intervals were estimated based on
the number of real clinical twin samples in the original data set that were classified as either chromosome Y
present or chromosome Y absent.
Table 18 Estimates for Trisomy 21, 18, and 13 in Simulated Population of Twin Pregnancies
Trisomy 21

Trisomy 18

Trisomy 13

Presence of Y

Sensitivity

96.4%

95.7%

93.6%

> 99.9%2-Sided 95% CI(86.4%, 98.9%)(68.3%, 99.4%)(64.1%, 98.9%)(99.9%, > 99.9%)

Specificity

99.9%> 99.9%> 99.9%> 99.9%2-Sided 95% CI(99.8%, > 99.9%)(99.9%, > 99.9%)(99.9%, > 99.9%)(99.7%, > 99.9%)

Table 18 provides point estimates and estimated 95% confidence intervals for the sensitivity and specificity of
VeriSeq NIPT Solution v2 to detect trisomy 21, 18, 13, and the presence of Y in a simulated population of twin
pregnancies consistent with the intended use population. Confidence intervals were estimated based on thenumber of QC passing clinical twin samples classified as either affected or unaffected by the relevant trisomy.
The sensitivity calculation assumes that two thirds of affected twin pregnancies are dizygotic with one affected
twin, while one third of affected twin pregnancies are monozygotic with both twins affected.
The estimates listed in Table 18 pertain to twin pregnancies only. Due to even lower prevalence, data for higherorder pregnancies (triplets or higher) were insufficient to establish appropriate statistical models to estimate
accuracy of aneuploidy detection.

Analytical Performance
Precision
To assess and quantify assay precision, a reanalysis of data using the VeriSeq NIPT Solution v2 analysis
pipeline software from two previous studies from the VeriSeq NIPT Solution was conducted:•

Multisite Reproducibility study that comprised three runs by three operators across three sites using asingle lot of reagent for a total of nine runs.

•

Within-Lab Precision study that comprised 12 runs at a single site using two ML STARs, two sequencing
instrument systems, and three sequencing reagent lots.

The objective of the precision study was to quantify the precision of the assay with regards to trisomy 21 (T21)and Chromosome Y and estimate variability among different instruments, library preparation kits, andsequencing reagent lots.
A 5% fetal fraction T21 pool was created by combining cfDNA extracted from maternal plasma from pregnant
women (with a T21 affected fetus) and cfDNA extracted from plasma from nonpregnant women. A 10% fetal
fraction maternal-male (XY fetus) cfDNA pool was also created. The sample panel for each study for each run
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VeriSeq NIPT Solution v2 Package Insert
included 4 replicates of the 5% fetal fraction T21 affected sample pool and 20 replicates of the 10% fetal
fraction maternal-male cfDNA pool. Testing was performed over 10 days for a total of 21 runs for the twostudies combined.
T21 and the presence of chromosome Y were chosen for evaluation based on representativeness of clinicalconditions and complexity of anomaly detection. As the smallest human autosome, the size of chromosome 21
has a direct impact on the sensitivity of T21 detection, particularly at low fetal fraction values such as thoseused in this study. Chromosome Y, as present in maternal plasma, is exclusively fetal in origin and therefore
easier for the assay to detect.
The observed mean and standard deviations for the Chromosome 21 LLR Score and the Chromosome Y
normalized chromosomal values (NCV) showed that replicate standard deviation (SD) was the largest source ofvariability. Variation between sites, instruments, and reagent lots added an insignificant amount of variability, as
evidenced by the difference between Total SD and Replicate SD in Table 19 and Table 20.
Table 19 Summary of Multisite (Reproducibility) Sequencing Response Standard Deviation (SD)ResponseN

Mean

Replicate SD

Total Reproducibility SD*

Chromosome 21 LLR Score

36

34.43

11.36

11.36

Chromosome Y NCV

180

190.56

7.96

10.20

* Total includes variability due to site, operator, run, day, and replicate.

Table 20 Summary of Within Lab Sequencing Response Precision
ResponseN

Mean

Replicate SD

Total Within Lab SD*

Chromosome 21 LLR Score

48

36.01

9.07

10.25

Chromosome Y NCV

240

198.68

7.63

7.82* Total includes variability due to sequencing instrument, reagent lot, operator, run, day, and replicate.

An additional study was performed to compare VeriSeq NIPT Solution v2 sequencing precision (total standard
deviation) using the version 2.0 of a flow cell versus version 2.5. The study included two types of flow cells(v2.0 and v2.5), three sequencing kit lots, four instrument systems, and two sequencing runs per combination
for a total of 48 runs at a single site. One sequencing pool was prepared from cfDNA plates that were manually
prepared. The sample panel included 4 replicates of the 5% fetal fraction T21 affected sample pool and 20
replicates of the 10% fetal fraction maternal-male (XY fetus) cfDNA pool. Results from the study are presented
in Table 21 and support that there is no difference in sequencing precision when using flow cell v2.0 versus flow
cell v2.5.
Table 21 Summary of Flow Cell v2.0 versus Flow Cell v2.5 Sequencing Response Precision
Response
Chromosome 21

Number of Observations

v2.0 Total

v2.5 Total

per Version

SD*

SD*

96

9.56

8.44

LLR Score

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Statistical Result**
Statistically Equivalent (pvalue=0.25)

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VeriSeq NIPT Solution v2 Package Insert

Response
Chromosome

Number of Observations

v2.0 Total

v2.5 Total

per Version

SD*

SD*

480

7.74

7.38

Y NCV

Statistical Result**
Statistically Equivalent (pvalue=0.38)* Total includes variability due to sequencing instrument, reagent lot, run, day, replicate**Based on F-test for equality of variances (standard deviations squared)

Cross-Contamination
Cross-contamination was assessed in the VeriSeq NIPT Solution sample preparation workflow. Plasma pools
from non-pregnant females (XX) and adult males (XY) were tested in a checkerboard pattern in the 96-wellplate format across 4 plates. N = 48 each for female and male samples per plate, for a total of 192 female and
192 male samples. None of the female samples demonstrated chromosome Y coverage that was statisticallyhigher than the estimated background, indicating no cross-contamination from male samples within the sameplate. No detectable cross-contamination was observed in the VeriSeq NIPT Solution.

Potentially Interfering Substances
The impact of potentially interfering substances was assessed in the VeriSeq NIPT Solution by evaluating theperformance of the assay in the presence of such substances.
Albumin, bilirubin, hemoglobin, and triglycerides (endogenous) were each spiked into maternal plasma pools
from unaffected female (XX fetus) pregnancies. They were tested at two concentrations for each test
substance (n=16 for each). No interference in the performance of the assay was observed.
Table 22 Potentially Interfering Substances (endogenous)
Test Substance

Low Test Concentration (mg/mL)

High Test Concentration (mg/mL)

Albumin

35

50

Bilirubin

0.01

0.15

Hemoglobin

100

200

Triglyceride

1.5

5Naturally occurring maternal genomic DNA (gDNA) in the plasma can also potentially interfere with assay
performance, as it can be extracted along with the fetal cfDNA. Genomic DNA levels at 1.6, 3.3, and 4.9 ng per
sample (corresponding to 1, 2, and 3 standard deviations above mean expected gDNA concentration after 7
days of whole blood storage12) was added to cfDNA extracted from maternal plasma from unaffected female(XX fetus) pregnancies. Samples were then tested in the VeriSeq NIPT Solution (n=16 for each concentration).
No interference in the performance of the assay was observed in the presence of elevated levels of gDNA.
Twenty drug-based potentially interfering substances (exogenous) commonly used or prescribed during
pregnancy were tested per EP7-A2 (Interference Testing in Clinical Chemistry; Approved Guideline-Second

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VeriSeq NIPT Solution v2 Package Insert
Edition). The 20 potential interferents were combined into four pools, spiked into maternal plasma from
unaffected female (XX fetus) pregnancies, and tested in the VeriSeq NIPT Solution (N=16 for each pool). No
interference in the performance of the assay was observed in the presence of these exogenous substances.
Table 23 Potentially Interfering Substances (exogenous)
Pool 1Pool 2Pool 3Pool 4

Acetaminophen

Diphenhydramine

Albuterol

Cetirizine

Acetylcysteine

Erythromycin

Bupropion

Dextromethorphan

Bisoprolol

Guaifenesin

Caffeine

L-Ascorbic acid

Citalopram

Heparin

Sertraline

Metoprolol

Desloratadine

Lidocaine

Sodium fluoride

Nadolol

Limit of Detection
The Limit of Detection (LOD) is defined as the level of fetal fraction that corresponds to the 95% detection
probability of a condition of interest, such as T21. To assess the LOD of VeriSeq NIPT Solution v2 for various
common conditions, studies and statistical analyses were conducted.
The probability of detection of a condition of interest in an affected sample processed by VeriSeq NIPT Solution
v2 primarily depends on three factors:•

fetal fraction•

sequencing depth•

size and complexity of the genomic region of interest.

Assuming constant sequencing depth, a given aberration is easier to detect in a sample with a higher fetal
fraction percentage than in a sample with a lower fetal fraction percentage. Conversely, assuming constantfetal fraction, a given aberration is easier to detect in a sample with a higher sequencing depth than in a sample
with a lower sequencing depth. Lastly, aberrations in smaller or more complex genomic regions are harder todetect than aberrations in larger or less complex genomic regions, assuming constant fetal fraction andsequencing depth.
To determine the LOD for T21 detection, samples comprising mixtures of pooled T21 samples and pooled
unaffected samples were analyzed. The two types of analyte were mixed over a titration series to create a setof seven fetal fraction levels (0, 2, 3, 4, 5, 6, and 10%). Each level was represented by a total of 10 replicates.
To further increase the resolution of the fetal fraction grid for the LOD analysis, the data from this study wasaugmented with data obtained from an in silico dilution. The effects of experimental dilution and titration were
simulated by the controlled mixing of sequencing data. The data from this in silico titration covered a set of 14
fetal fraction levels (1.25, 1.50, 1.75, 2.00, 2.25, 2.50, 2.75, 3.00, 3.25, 3.50, 3.75, 4.00, 4.25, and 4.50%) with
32 replicates for each level. A probit analysis was applied to the resulting data to determine the LOD for T21.

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Independently, a statistical model using fetal fraction, sequencing depth, and genomic size/complexity wasdeveloped to predict the detection probability of any aberration in any sample. This model was established fromthe data corresponding to a set of 1,405 XY samples. The LOD for T21, as predicted by this model, wasdetermined to be concordant with the probit-based estimate described above. This statistical model was used
to estimate LOD values for aneuploidies on all autosomes and for partial deletions and duplications.
Figure 2 shows the 95% detection probability for average regions by size and the autosomal limits of detection
for all trisomies and all monosomies.
Figure 2 95% Detection Probabilities for Average Regions by Size for the VeriSeq NIPT Solution v2

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VeriSeq NIPT Solution v2 Troubleshooting
Failure Mode

Possible
ResultInterpretation

Recommended Action

Comments

Redraw

Based on visual inspection of plasma

Insufficient

Sample QC

Insufficient

input plasma

failureplasma volume.

Blood tube

Blood is not

Sample was not

Make sure that the

failureseparated into

centrifuged.

centrifuge started andlayersvolume.

the tube was spun at thecorrect force. Redraw
sample.
Improper sample
storage or

Frozen samples will not separate.
Improper transport or storage conditionstransport

might lead to hemolysis of samples.

(hemolysis ofsample).

Redraw sample.

VeriSeq NIPT Solution v2 Package Insert

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Troubleshooting

58

Possible
ResultInterpretation

Recommended Action

Sample clog or

PlasmaIndividual

Inspect sample. If

slow flow

contamination

samples can clog

remaining plasma inthe binding platetube is red or milky,

if there iscancel sample andsignificant

request redraw. If

contamination insample appears normal,

the plasma

retest sample.

Comments

sample.
SampleInadequate visual

Invalidate any samples in Might indicate that samples were

overflow

inspection of eachnearby wells affected bytransported or stored improperly before

tube for sample

the overflow.

processing. Exclude unsuitable samples

suitability.

from processing.

HardwareInadequate

Retest sample. If

malfunction

digestion ofproblem persists in wellmaterial duringlocation with otherextraction.

samples, contact
Illumina TechnicalSupport.

VeriSeq NIPT Solution v2 Package Insert

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Failure Mode

59

Possible
ResultInterpretation

Recommended Action

Comments

Individual

Sequencing

Possible causes

Check Sample

Indicates either an insufficient sample

SampleQC failure

are as follows:• Insufficient

Annotation. Check for

input or a mistransfer on the ML STAR.

similar performance on

Insufficient genetic material can come fromgenetic input• Mistransfer

previous samples ininsufficient cell-free DNA in the plasma or

relative plate position.

cell-based DNA causing overdilution of theRetest sample.

sample for sequencing.

Analysis QC
failureduring sample
handling• Sequencing
reagent failure
Low FF or non-Insufficient data

Retest from plasma.

excluded sites

generated to(NES) countaccurately report.

Quantification

FailedInsufficient

Repeat quantification. If

Non-passing standard curve metrics either

QC failure

quantification

process yield.

repeat fails, contact

indicate issues with library preparation (i.e.

run. Batch

Illumina Technicaluse of non biological grade ethanol) or

median below

Support.

issues with the quantification process.

minimum
FailedStandard curve

Repeat quantification. If

quantification

failure.

repeat fails, contactrun

Illumina TechnicalSupport.

Pooling failure

Failure to

Pooling analysis

Reevaluate target poolcompleteis unable toconcentration. Rerun

sample pooling

calculate proper

pooling analysis.

pool volumes.

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Failure Mode60

Process
Step
Batch

Error CodeError Dialog

Description

User Resolution

EM0044The Batch ID

The VeriSeq NIPT Solution v2

Rename batch using a name that does notenteredonly accepts numbers, letters,

contain special characters.

contains

underscores, and dashes forforbidden

all data fields.

Creationcharacters.
Batch

EM0051Creation

Batch

EM0076Creation

The Batch ID is

The VeriSeq NIPT Solution v2

Rename batch using a name that is fewer thangreater than 36

limits the length of batch

36 characters.

characters innames to 36 characters or

length.

less.

Unable to

The VeriSeq Onsite Server v2

1. Make sure that the ML STAR is connected toconnect tois not responding to data

VeriSeq Onsite

requests from the Workflow

the network.
2. Make sure that the VeriSeq Onsite Server v2

Server v2

Manager.

is on.
3. Check that the ML STAR can connect to the
VeriSeq Onsite Server v2 (via ping request).
4. Check the vacuum waste bottle. If the waste
bottle is more than half full, empty the waste
bottle.
5. If previous steps do not resolve the issue,
contact Illumina Technical Support.

Batch
Creation

EM0118This batch has

The specified batch has

The batch record on the VeriSeq Onsite Server

been failed andalready failed and cannot be

v2 indicates that the selected batch has failed.

cannot beprocessed any further.

No further processing is allowed. Create

further
processed.

another batch with the required samples.

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61

Error CodeError Dialog

Description

User Resolution

Batch

Not

This batch has

The indicated batch has

Creation

applicablealready

processed through pooling.

completed

The only allowable processing

Repool as follows.
• Select Re-Pool.
• Abort method and ensure batch name isprocessing.

is repool.

Stepcorrect before repooling.

Would you liketo repool?
Plasma

WP0087Isolation

Duplicate

Samples with identical

1. Follow the Workflow Manager prompts tosamplebarcodes have been loaded

barcodesonto the system.

identify which samples are duplicates.
2. Remove the duplicates and relabel orreplace them.
3. Reload the samples.

loaded.
PlasmaEP0102Isolation

Samples

Samples included in the

1. Follow the Workflow Manager prompts tospecified in thesample sheet were not

Sample Sheet

included in the loaded

were notbarcodes.

identify missing samples.
2. Do one of the following options:• Add missing samples to the batch and reload
samples.
• Abort method, modify the sample sheet as

loaded.

needed. Restart method.
Plate Load

Not

Venus Barcode

The Workflow Manager

1. Check plate placement to confirm that theapplicable

Mask Error

enforces correct plate-tobatch association using Venus

plate layout is correct.
2. Make sure that the plate loaded is thebarcode masks.

correct plate for the indicated batch.

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Step
cfDNA

Error CodeError Dialog

Description

User Resolution

WE0150Pressure in the

The Workflow Manager does

1. Check for kinks or other obstructions in thevacuumnot proceed if the resting

chamber is too

vacuum line pressure sense isvacuum line.
2. Open the waste line release clips, allow for

low.

< 400 Torr.

pressure to release, and then fully close theExtraction

release line clips.
3. Make sure that the vacuum controller andpump are turned on.
4. If the problem persists, contact Illumina
Technical Support.
cfDNA
Extraction

WE0153Pressure in the

If the measured vacuum

On the back of the controller, make sure that all

vacuumpressure is too high before thevacuum connections and lines are secure.

chamber is toopressure control starts, thehigh.

system might bemalfunctioning.

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Step
cfDNA

Error CodeError Dialog

Description

User Resolution

WE0996

Vacuum failed

The seal failure must be

Verify seal failure is resolved prior to selecting

to seal.

resolved before continuing.

OK .
1. Make sure that the binding plate is flush

Extraction

against the vacuum manifold. With a gloved
hand, forcibly press down on the binding plate.
2. Listen for the vacuum hum and observe theflow of water through the binding plate.
3. Open the trace view on the Workflow
Manager. After the actual pressure reading
reaches at least 50 pressure units less than theambient reading, select OK to continue with
cfDNA Extraction.
4. If the required pressure reading is notreached during the allotted time, select OK tocontinue with the first lysate load.
5. Pause the method after the lysate isdispensed onto the binding plate. Reseat andforcibly press down on the binding plate.
6. If the lysate fails to flow through the plate,contact Illumina Technical Support.
cfDNA
Extraction

WM0219If Vacuum is on,

The vacuum might stay on

1. On the Vacuum Controller, press the Powermanually rest

after a method abort during

the pump.

extraction.

button to shut off the vacuum.
2. Wait 10 seconds, and then press the Powerbutton again to turn on the vacuum.

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Step
cfDNA

Error CodeError Dialog

Description

User Resolution

EE0477

An error has

If an iSWAP error isMake sure that the plate can be recovered (nooccurred while

encountered (plate drop,

moving a plate.
(iSWAP error)

failed to pick up, etc), thespilled material).
• If the plate cannot be recovered, abort theExtraction

system prompts you tocomplete the plate move
manually.

run.
• If the plate can be recovered, follow thedisplayed instructions to complete the platetransfer manually.

cfDNA
Extraction

EE0519Scanned

The loaded Binding plate does

Make sure that the plate being loaded matches

barcode does

not match the barcode of thethe recorded barcode (see the trace log for the

not matchplate removed.

expected barcode).

Binding Platebarcode onrecord.

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Step
API

Error CodeError Dialog

Description

EA0372

Unable to

The VeriSeq Onsite Server v2

connect to theis not responding to data

1. Make sure that the ML STAR is connected todata server.

requests from the Workflow

the network.
2. Make sure that the VeriSeq Onsite Server v2

Manager.

User Resolution

is on.
3. Check that the ML STAR can connect to the
VeriSeq Onsite Server v2 (via ping request).
EA0774

Connection

The VeriSeq Onsite Server v2Error.
The API server

has stopped responding toconnection

Workflow Manager.

data requests from theMake sure that:
1. Make sure that the ML STAR is connected to
the network.
2. Check that the ML STAR can connect to the
VeriSeq Onsite Server v2 (via ping request).
3. Make sure that the VeriSeq Onsite Server v2

failed tovalidate.

is on.
EA0780403: Invalid

The data sent violates theConsult the error details for more information.

RequestThe current

system workflow logic.

Common causes involve inputs that are tootransaction is
not valid.

long or violate the acceptable character list.

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References
1.

Nagaoka S, Hassold T, Hunt P. Human aneuploidy: mechanisms and new insights into an age-old
problem. Nat Rev Genet. 2012;13(7):493-504. doi:10.1038/nrg3245.

2. Garnder RJ, Sutherland GR, Schaffer LG. Chromosome Abnormalities and Genetic Counseling. 4thedition. New York (NY): Oxford University Press; 2012.
3. Akolekar R, Beta J, Picciarelli G, Ogilvie C, D'Antonio F. Procedure-related risk of miscarriage
following amniocentesis and chorionic villus sampling: a systematic review and meta-analysis.
Ultrasound Obstet Gynecol. 2015 Jan;45(1):16-26. doi: 10.1002/uog.14636.
4. American College of Obstetricians and Gynecologists. Screening for fetal aneuploidy. Practice
Bulletin No. 163. Obstet Gynecol. 2016; 127(5):e123-137.
5. Gil MM, Accurti V, Santacruz B, Plana MN, Nicolaides KH. Analysis of cell-free DNA in maternal bloodin screening for fetal aneuploidies: updated meta-analysis. Ultrasound Obstet Gynecol. 2017 Apr 11.
doi: 10.1002/uog.17484.
6. Bianchi D, Parker R, Wentworth J et al. DNA Sequencing versus Standard Prenatal Aneuploidy
Screening. N Engl J Med. 2014;370(9):799-808. doi:10.1056/nejmoa1311037.
7. Benn P, Borrell A, Chiu RW, et al. "Position statement from the Chromosome Abnormality Screening
Committee on behalf of the Board of the International Society for Prenatal Diagnosis." Prenat Diagn
35 (2015): 725-34.
8. Gregg AR, Skotko BG, Benkendorf JL, et al. Noninvasive prenatal screening for fetal aneuploidy,
2016 update: a position statement of the American College of Medical Genetics and Genomics.
Genet Med. 2016: doi:10.1038/gim.2016.97.
9. Dondorp W, de Wert G, Bombard Y, et al. Non-invasive prenatal testing for aneuploidy and beyond:challenges of responsible innovation in prenatal screening. Eur J Hum Genet. 2015 Nov;23(11):143850.
10. Grati, et al. "Fetoplacental mosaicism: potential implications for false-positive and false-negative
noninvasive prenatal screening results." Genetics in Medicine 16 (2014): 620–624.
11. Wellesley, et al. "Rare chromosome abnormalities, prevalence and prenatal diagnosis rates from
population-based congenital anomaly registers in Europe." European Journal of Human Genetics 20(2012): 521-526.
12. Norton S, Lechner J, Williams T, Fernando M et al. A Stabilizing Reagent Prevents Cell-free DNA
Contamination by Cellular DNA in Plasma During Blood Sample Storage and Shipping as Determinedby Digital PCR. Clin.l Biochem. 2013;46: 1561–1565. doi: 10.1016/j.clinbiochem.2013.06.002.
13. Bianchi D W, et al. "Genome-wide fetal aneuploidy detection by maternal plasma DNA sequencing."
Obstet Gynecol 119 (2012): 890-901.
14. Ehrich M, et al. “Genome-wide cfDNA screening: clinical laboratory experience with the first 10,000
cases.” Genet Med 19 (2017): 1332-1337.

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VeriSeq NIPT Solution v2 Package Insert

15. Fiorentino F, et al. “The clinical utility of genome-wide cfDNA screening.” Prenat Diagn 37 (2017):
593-601.
16. Pertile, MD, et al. "Rare autosomal trisomies, revealed by maternal plasma DNA sequencing, suggest
increased risk of feto-placental disease." Sci Transl Med 9 (2017): eaan1240.

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Revision History
Document

Date

Document #August 2022

Update the workflow part number
Removed instruction to pipette to mix if library plate was frozen.

May 2022

Split Limitations of the Procedure into VeriSeq NIPT Solution v2

1000000078751 v08
Document #1000000078751 v07

Description of Change

Reporting and include first two bullets. Remaining text in a newheader Limitations of the Assay.
Removed• VeriSeq from all reagent labeling.
• Apply a plate barcode to VeriSeq NIPT Adapter Plate in
Prepare Libraries preparation.
Added• The word certified to DNase/RNase-free water.
• One of the following microplate readers, or equivalent andSpectraMax M2, M3, M4, M5, and note.
• To the VeriSeq NIPT Microlab STAR section to explain what to
do during an error handling event.
• A note to visually inspect wells.
• Instructions for 24 and 48-sample batches throughout
protocol sections.
• Steps for when to use the purple adapter plate or equivalent.
• Verbiage to Demographics and Pregnancy Characteristics
section to include first trimester pregnancy results.
• A bullet to the Deep Well plate specifications to include
torque-resistant.
Updated• Verbiage for unique batch names for clarity and include an
example.
• Symbols and formatting for Notes, Cautions, and Warnings.
• Results of the test subbullets.
• Guanidine thiocyanate to guanidine hydrochloride.
• CVS to BVS (Basic Vacuum System)• Verbiage for using the genomewide screen and the LLR score.
• Specs: Reagent tub specs, deep-well plates, 384 well plates,
96 well plates

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VeriSeq NIPT Solution v2 Package Insert
Document

Date

Description of Change

Document #August 2021

Updated EU Authorized Representative address.

DecemberUpdated Principles of Procedure, Warnings and Precautions,2020

and Product Labeling sections with additional clarifications to1000000078751 v06
Document #1000000078751 v05

fulfill regulatory requests.
Minor updates to content in protocol to match current Illumina
style and organization.
Corrected description of chromosome 21 as "second smallest
human autosome" to "smallest human autosome" in Precisionsection of Analytic Performance.
Added caution statements to address improper use ofreservoirs and risks of sample amalgamation to Isolate Plasma
Preparation and Interpretation of Results sections.
Added new server and software part numbers for the release of
new server model and software part number updates.
Added cautions to protocol and troubleshooting information foraddressing and preventing sample overflows.
Updated active ingredients in the reagent DNA Quantification
Standard in the Accessory Box to align with the Safety Data
Sheet.
Updated naming conventions of the Local Run Manager VeriSeq
NIPT module for consistency with other documentation.
Added revision history.
Document #October

1000000078751 v04
Document #Minor corrections.

2020
September

1000000078751 v03

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2020

Updated materials list to present specifications of labware alongwith known compatible options.

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VeriSeq NIPT Solution v2 Package Insert
Document

Date

Document #February

Updated presentation of Clinical Performance information to2020

better convey the differences between basic and genomewide

1000000078751 v02

Description of Change

screen types.
Added new Differences in Performance Between the Basic andGenomewide Screens section.
Removed contradictory information about the optionality of the
supplementary report from the Principles of Procedure section.
Updated naming convention of the VeriSeq NIPT Workflow
Manager v2 software throughout the document for stylistic
consistency.
Updated labeling of Australia and Illumina Netherlands
addresses to reflect recent changes.
Document #August 2019

1000000078751 v01
Document #Removed duplicate step in Extract cfDNA caused by publishingsoftware error.

May 2019

Initial release.

1000000078751 v00

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VeriSeq NIPT Solution v2 Package Insert
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© 2022 Illumina, Inc. All rights reserved.
All trademarks are the property of Illumina, Inc. or their respective owners. For specific trademark information, see
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Contact Information
Illumina
5200 Illumina Way

Australian Sponsor

San Diego, California 92122 U.S.A.

Illumina Australia Pty Ltd+1.800.809.ILMN (4566)

Nursing Association Building+1.858.202.4566 (outside North America)

Level 3, 535 Elizabeth Street

techsupport@illumina.com
www.illumina.com

Illumina Netherlands B.V.
Steenoven 19

Melbourne, VIC 3000
Australia

5626 DK Eindhoven
The Netherlands

Product Labeling
For a complete reference of symbols that appear on product packaging and labeling, refer to the symbol key atsupport.illumina.com on the Documentation tab for your kit.
A Summary of Safety and Performance (SSP) is located at https://ec.europa.eu/tools/eudamed, after launch of
the European Database on Medical Devices (Eudamed). It is linked to the Basic UDI-DI (0081627002NIPTRP).

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