nCounter Vantage 3D SNV:Fusions Lung Assay That Count® 3 NanoString Technologies® nCounter® Vantage 3D™ SNV:Fusions Lung Assay for MAX and FLEX Analysis Systems nCounter Vantage

Molecules That Count® MAN-10038-02 November 2016

nCounter® Vantage 3D™ SNV:Fusions Lung Assay

for MAX and FLEX Analysis Systems

Protocol

NanoString Technologies, Inc.

530 Fairview Ave North

Seattle, Washington 98109 USA

Telephone: 206.378.6266

888.358.6266

E-mail: [email protected]

http://www.nanostring.com/

mailto:[email protected]

2

NanoString Technologies® nCounter® Vantage 3D™ SNV:Fusions Lung Assay for MAX and FLEX Analysis Systems

FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.

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This nCounter Vantage 3D SNV:Fusions Lung Assay for MAX and FLEX Analysis Systems and its contents are the

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Subject to the terms and conditions of sale of the nCounter Analysis System, NanoString grants you a limited, non-exclusive, non-transferable, non-sublicensable, research use only license to use this proprietary nSolver™ software with the nCounter Analysis System only in accordance with this manual, the manual for the nCounter Analysis System, and other written instructions provided by NanoString. Except as expressly set forth in the terms and conditions, no right or license, whether express, implied, or statutory, is granted by NanoString under any intellectual property right owned by or licensed to NanoString by virtue of the supply of this software or the proprietary nCounter Analysis System. Without limiting the foregoing, no right or license, whether express, implied, or statutory, is granted by NanoString to use the nSolver Analysis Software or nCounter Analysis System with any third party product not supplied or licensed to you by NanoString, or recommended for use by NanoString in a manual or other written instruction provided by NanoString.

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NanoString Technologies, NanoString, nCounter, Vantage 3D, nSolver, nCounter MAX, nCounter FLEX, 3D Biology and Molecules That Count are trademarks or registered trademarks of NanoString Technologies, Inc., in the United States and/or other countries. All other trademarks and/or service marks not owned by NanoString that appear in this document are the property of their respective owners.

Copyright

© 2016 NanoString Technologies, Inc. All rights reserved.

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Molecules That Count® 3


nCounter Vantage 3D Lung SNV:Fusions Lung Assay for MAX and FLEX Analysis Systems

Overview

In this nCounter Vantage 3D SNV:Fusions Lung Assay, the nCounter Vantage 3D DNA SNV Solid Tumor

Panel is combined with the nCounter Vantage Lung Gene Fusion Panel in a 3D Biology™ workflow (see

FIGURE 1) to permit simultaneous detection of fusion transcripts that are associated with lung cancer

from RNA and detection of more than 100 solid-tumor associated single nucleotide variants (SNVs),

multinucleotide variants (MNVs), and insertion/deletion variants (InDels).

The nCounter Vantage 3D DNA SNV Solid Tumor Panel enables detection of sequence variants from

human genomic DNA that has undergone target enrichment by multiplex PCR. This is enabled by a

modified version of the nCounter TagSet chemistry (see FIGURE 2). Each two-arm Probe S is formed by

two oligonucleotides hybridized together through a unique stem sequence to create a probe with two

distinct binding regions. The Probe S Pool contains a pair of S probes for every variant that is assayed. One

S probe is designed to detect the presence of the variant sequence and the second S probe is designed to

report on the presence of the reference sequence.

Since reference sequences predominate in most samples for most positions tested, the total molecular

counts on reference targets are attenuated by adding competitive Reporter binding oligonucleotides

referred to as M probes (not shown in FIGURE 2). T probes serve to increase target specificity and facilitate

detection on nCounter systems. All S and T probes have been designed to target sequences as defined by

the GRCh37/hg19 assembly of the human genome. A single nucleotide mismatch between the S probe

and the target in either binding region of the S probe can disrupt probe hybridization to the target, thereby

providing single-nucleotide specificity for each probe. The panel is designed to provide data that upon

analysis yields positive detection calls for DNA mutations and variant alleles associated with solid tumor

biology that are present at an allele frequency of 5% or greater.

The nCounter Lung Gene Fusion Panel includes 63 probes: 35 for specific fusion detection, 24 for

positional gene expression imbalance detection, and 4 internal reference genes. The nCounter Vantage

3D Lung Gene Fusion Panel is designed to provide sensitive and specific detection of 35 lung-cancer

associated gene fusions and positional gene expression imbalance detection for the genes ALK, RET, and

ROS1 from purified total RNA and RNA extracted from FFPE samples. This panel utilizes nCounter TagSet

chemistry with the addition of a Protector Probe, an oligonucleotide that is complementary to a portion

of Probe A, allowing for a new level of target specificity to be created (see FIGURE 3). Probe A, which

links the unique reporter tag to a specific target sequence, recognizes both halves of the exon-exon

junction. To minimize off-target hybridization, Probe A is made partially double-stranded by the addition

of the Protector oligonucleotide to the hybridization mix. This combination of Probe A and the Protector

Probe (also called Probe P) is referred to as a junction probe.

Contact NanoString Support ([email protected]) to receive additional assistance with this assay.


4


Figure 1. The nCounter Vantage 3D SNV:Fusions Lung Assay workflow



Figure 2. The nCounter Vantage 3D DNA SNV Solid Tumor Panel probe chemistry. As shown, a single nucleotide mismatch will

disrupt Probe S / target hybridization, yielding single nucleotide specificity for detection of sequence variants. Only the presence

of exact sequences of interest result in a significant number of Reporter binding events.

Figure 3. Junction probe design methodology. Junction probes span a unique fusion junction, using toehold exchange technology

for greater specificity. For a technical explanation of toehold exchange technology, see Zhang DY, Chen SX, Yin P. (2012)

Optimizing the specificity of nucleic acid hybridization. Nat Chem 4(3):208-14.

6


System Qualification

SNV panels can only be successfully run on nCounter MAX™ or nCounter FLEX™ analysis systems that

have been performance-qualified for this panel type. SNV panels cannot be used on nCounter SPRINT™

Profiler analysis systems. Please consult the SNV Qualification Kit Manual (MAN-10039) for more details

about how to obtain and use the materials needed for this system testing as well as how to

communicate the results of the testing to NanoString Support ([email protected]). The SNV

Qualification Kit process must be completed prior to running the SNV:Fusions Lung Assay for the first

time. Contact NanoString Support ([email protected]) to receive additional assistance with the

system qualification.

Reference Run

Prior to running the SNV:Fusions Lung Assay with experimental samples, it is necessary to collect one full

cartridge (12 lanes) of DNA SNV Reference Sample data. NanoString will provide one extra nCounter

Vantage 3D SNV Kit to be used for the reference run. Please do not use this kit for any other purposes.

Please see Appendix: DNA SNV Reference Run for details.





Materials and Reagents

Table 1. Core materials provided by NanoString for the DNA SNV Solid Tumor Panel

*The DNA SNV Solid Tumor Panel is designed to be compatible with other nCounter Vantage RNA and protein panels. The

nCounter Master kit must be purchased separately.

**The DNA SNV Reference Sample is obtained from the US National Institute of Standards & Technology (NIST). It is Reference

Material 8398: Human DNA for Whole-Genome Variant Assessment. It is homozygous for reference alleles at every position that

is assayed by the DNA SNV Solid Tumor Panel and serves as a negative control and reference sample for each cartridge run.

Table 2. Core materials provided by NanoString for use of the Lung Gene Fusions Assay (for each hybridization

reaction)

Reagents Volume (µL) Description

nCounter TagSet 28 TagSet for fusion detection

Probe A Pool 3 Probe A for fusion junction detection

Probe B Pool 3 Probe B for fusion junction detection

Probe P Pool 3 Protector probe to increase specificity of Probe A

Item Reagents Volume (µL) Description

nCounter Vantage 3D

DNA SNV Solid Tumor

Panel*

SNV Solid Tumor Primer Pool

18 Primers for DNA amplification

5X dU Amp Master Mix (also referred to as dU-containing Amp Master Mix)

28 Master Mix for DNA amplification, with dUTP and UDG to prevent carryover contamination, 5X concentration

SNV TagSet 28 TagSet for SNV detection

SNV Solid Tumor Probe M Pool

16.5 Oligonucleotides to attenuate reference allele sensitivity

SNV Solid Tumor Probe S Pool

3 Probe S pool for SNV detection

SNV Solid Tumor Probe T Pool

3 Probe T pool for SNV detection

DNA SNV Reference Sample

32 Human Genomic DNA reference

sample (2.5 ng/L) for up to 12 reactions. To be used in the DNA SNV Reference Run and in lane 12 of all other reactions.

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Table 3. Equipment required for use of the SNV:Fusions Lung Assay

*See Thermal Cycler Guidelines below.

**We recommend using a Qubit Fluorometer for RNA and DNA quantification.

Thermal Cycler Guidelines

Please note that a thermal cycler that has a heated lid is required for this protocol. Thermal cyclers are

produced by a wide variety of manufacturers and possess a wide variety of features. We recommend

using a model with a programmable heated lid. Models without programmable lids may reach a very

high temperature that causes tubes to melt or deform. However, programmable lids may offer different

levels of control.

Ideally, NanoString recommends a thermal cycler with a heated lid that can adjust throughout the protocol. The heated lid should be set to 5° C greater than the current incubation temperature at any moment.

Some heated lids cannot be assigned a floating temperature. In this situation, program the heated lid to be 5° C greater than the maximum temperature during the protocol. The heated lid should not exceed 110° C.

Table 4. Optional materials for use of the DNA SNV Tumor Panel

*NanoString recommends using only genomic DNA Reference Standards from Horizon Discovery, plc as positive controls. These

standards are typically blends of genomic DNA purified from engineered human cell-lines that have one or more sequence

variants, occurring at precisely verified allele frequencies, which can be assayed by the nCounter Vantage 3D DNA SNV Solid

Tumor Panel. For example, The Tru-Q1 Reference Standard (Catalog ID HD728) features several BRAF, EGFR, and KRAS variants

at somatic allele frequencies.

Instruments

NanoString nCounter FLEX or nCounter MAX analysis system

Microcentrifuge or picofuge

Pipettes (p1000, p100, p20, p10)

Calibrated thermal cycler with a heated lid*

Qubit™ Fluorometer**

Material Part Manufacturer

Positive control gDNA* Various Horizon Discovery



Table 5. Additional materials required for use of the SNV:Fusions Lung Assay (not provided)

*Equivalent kits may be substituted if they offer similar function and reliability.

**Master Kit part numbers vary based on the number of reactions ordered. Item # 100052 (12 reactions), 100054 (48 reactions) and 100050 (192 reactions).

Table 6. Optional equipment for use of the SNV:Fusions Lung Assay

*The peak of the fragmented DNA profile should be greater than 200 bp.

**At least 50% of the RNA sample should be greater than 300 nucleotides.

Material Part Manufacturer

Nuclease-free water Various Various

TE buffer, pH 8.0 Various Various

AllPrep DNA/RNA Mini Kit* (for use with fresh/frozen samples)

80204 QIAGEN

AllPrep DNA/RNA FFPE Kit* (for use with FFPE samples)

80234 QIAGEN

Deparaffinization Solution (for use with the AllPrep FFPE Kit)

19093 QIAGEN

Thin-wall PCR strip tubes Various Various

1.7 mL microcentrifuge tubes Various Various

Qubit dsDNA HS Assay Kit Various ThermoFisher

nCounter Master Kit Various** NanoString Technologies

Instrument

Agilent TapeStation or Bioanalyzer or other DNA* and RNA** fragment analysis instrumentation

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RNA Sample Preparation

1. If using formalin-fixed, paraffin-embedded (FFPE) samples, extract RNA using standard protocols

outlined in the AllPrep DNA/RNA FFPE Kit or by using a comparable method.

Evaluate RNA quality using a fragment analysis system to measure nucleic acid fragmentation.

We recommend that at least 50% of the sample be greater than 300 nucleotides in length for

optimal performance.

2. If using fresh/frozen samples, extract RNA using standard protocols outlined in the AllPrep DNA/RNA

Mini Kit or by using a comparable method.

3. Quantify RNA concentration using a Qubit RNA HS Assay Kit and Qubit Fluorometer or a comparable

fluorescence based method. Quantification by spectrophotometry, as with a NanoDrop instrument

for example, is prone to provide inaccurate and overestimated measures of RNA concentration.

4. 50-100 ng of total RNA purified from fresh or frozen tissue or 150-300 ng of RNA purified from FFPE

sample is required for each reaction.



DNA Sample Preparation

1. The DNA SNV Reference Sample provided by NanoString is highly purified; it does not require

further purification prior to use.

2. If using fresh/frozen samples, extract DNA using standard protocols outlined in the QIAGEN DNeasy

Blood & Tissue Kit or by using a comparable method or kit.

3. If using formalin-fixed paraffin-embedded (FFPE) samples, extract DNA using standard protocols

outlined in the AllPrep DNA/RNA FFPE Kit or by using a comparable method or kit.

The DNA SNV Solid Tumor Panel requires a multiplex PCR enrichment step that uses amplicons

that range from 150 to 200 bp in length. In general, FFPE-extracted gDNA is less amplifiable than

gDNA obtained from fresh or frozen tissue samples. As described in step 11 of DNA Sample

Amplification, to compensate for this, 2 additional cycles (from 18 cycles to 20 cycles) of

multiplex PCR are recommended for FFPE-extracted gDNA as are needed for fresh/frozen

sample-derived gDNA.

Successful PCR enrichment has been accomplished from a wide range of FFPE samples of

differing apparent qualities and a number of methods to estimate the quality of FFPE-derived

gDNA exist. For example, FFPE-derived human gDNA with DNA Integrity Numbers (DIN, obtained

with an Agilent TapeStation Instrument) ranging from 1.7 to 6.9 have all yielded accurate SNV

detection from the workflow. However, samples with a DIN of 3 or lower may require 21 cycles

of multiplex PCR; samples of this low quality are less likely to yield reliable results. Other

methods to determine the quality of FFPE-derived gDNA use qPCR methods to measure the

relative amplifiability of the DNA.

If you choose to evaluate the DNA quality of your samples prior to use of the DNA SNV Solid

Tumor Panel, choose a method or kit that measures whether 200 bp amplicons can be robustly

amplified.

4. Quantify DNA concentration using a Qubit dsDNA HS Assay Kit and Qubit Fluorometer or a

comparable PicoGreen® fluorescence based method. Exactly 5 ng of DNA is required for each

reaction. Quantification by spectrophotometry, as with a NanoDrop instrument, for example, is

prone to provide inaccurate and overestimated measures of DNA concentration.

5. When using the DNA SNV Solid Tumor Panel, variant calling accuracy and precision are optimized by

running the DNA SNV Reference Sample from Materials and Reagents (Table 1) in lane 12 on each

cartridge (provided in the nCounter Master Kit).

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DNA Sample Amplification

Each amplification reaction will contain the following components:

Table 7. DNA sample amplification reaction components

Component Volume (L)

5X dU Amp Master Mix 2

SNV Solid Tumor Primer Pool 1

Sample DNA 5 ng*

(Total volume should not exceed 5 L)

Nuclease-free Water Up to the final volume noted below

Total Volume 10

*The minimum concentration of the DNA sample should be 1 ng/µL.

IMPORTANT: The following protocol has been optimized for a fixed sample DNA mass input of 5 ng.

Accurately measure all sample DNA with a fluorescence-based method (e.g., a Qubit dsDNA HS Assay

Kit) and only use 5 ng input with the DNA SNV Solid Tumor Panel.

1. Remove tubes of the SNV Solid Tumor Primer Pool and the 5X dU Amp Master Mix from the

freezer and thaw on ice.

2. Thaw the SNV DNA Reference Sample from Materials and Reagents (Table 1) and the prepared

DNA samples from DNA Sample Preparation (if previously frozen) and chill nuclease-free water

on ice. Invert or flick tubes several times to mix well and briefly spin down reagents.

3. On ice, create a Reaction Mix sufficient for 14 reactions by adding the following to a clean microfuge tube:

14 μL of SNV Solid Tumor Primer Pool

28 μL of 5X dU Amp Master Mix

Mix by gentle pipetting six or more times while taking care not to produce bubbles, then briefly

spin down.

4. Label 12-strip PCR tube(s). Ensure that the tubes will fit in a microfuge or picofuge.

5. Add 3 μL of Reaction Mix from Step 3 to each of the 12 tubes. Use a fresh tip for each pipetting

step to accurately pipette the correct volume.

6. Add 5 ng of DNA in nuclease-free water or TE buffer (minimum concentration 1 ng/μL) to each tube.

NOTE: Make sure that the tube that will correspond to lane 12 is loaded with 5 ng of the DNA SNV Reference Sample that is provided with the panel.



7. If necessary, add nuclease-free water to bring each amplification reaction to

10 μL.

8. Mix by gentle pipetting six or more times while taking care not to produce bubbles and then briefly spin down.

9. Place the PCR 12-well strip tube(s) in a thermal cycler.

NOTE: If space is available, label clearly and cut each 12-well strip tube in half. Place both halves

in the middle of the heat-block. This will reduce the potential for edge effects that are

characteristic of some under-performing thermal cyclers.

10. Run the following protocol:

Table 8. DNA Amplification Thermal Cycler Protocol

Step Temperature Time

UDG digest 37 ˚C 30 min

UDG inactivation 50 ˚C 5 min

Initial denature 95 ˚C 3 min

18-21 cycles*

Denature 95 ˚C 15 sec

Anneal 56 ˚C 2 min

Extension 68 ˚C 30 sec

Final extension 68 ˚C 5 min

Hold 4 ˚C Hold

IMPORTANT: Immediately prior to assembling the hybridization reactions (described in DNA

SNV Hybridization), the amplified DNA must be heat-denatured then quick-chilled as described

in steps 11 and 12 below.

11. After PCR cycling, denature the DNA in each tube at 95 ˚C for 10 minutes.

12. After heat-denaturation, immediately place the DNA on ice to rapidly cool it and to minimize any reannealing that may otherwise occur. Heat denaturation followed by ramping down to 4 ˚C in the thermal cycler does NOT cool the DNA rapidly enough to minimize reannealing. Leave the amplified DNA on ice for at least 5 minutes.

*IMPORTANT: DNA extracted from formalin-fixed paraffin-embedded (FFPE) samples is typically degraded due to the fixation and storage process. To ensure sufficient amplified DNA is produced, the number of PCR cycles should be increased from the recommended 18 cycles. For FFPE-extracted DNA use 20 cycles by default. Some particularly degraded samples may require 21 cycles (see Table 8). For accurate variant detection, do not exceed 21 cycles of amplification.

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DNA SNV Hybridization

GENERAL PROBE HANDLING INSTRUCTIONS:

During setup, do not vortex or pipette vigorously to mix.

Mixing should be done by flicking or inverting the tubes.

If using a microfuge to spin down tubes, do not spin any faster than 1,000 RCF for more than 30 seconds.

Do not “pulse” to spin because that will cause the centrifuge to go to the maximum speed and may spin the TagSet out of solution.

Each hybridization reaction will contain the following components:

IMPORTANT: Instructions for preparing the Probe S Pool, Probe T Pool, and the Probe M Pool working

stocks are provided in steps 3–5 below. When making these working stocks, mix gently but thoroughly,

such as by flicking the tubes or performing several cycles of up-and-down pipetting to ensure obtaining

homogeneous working solutions.

Each DNA SNV hybridization reaction will contain the following components:

Table 9. DNA SNV hybridization reaction components

Component Volume (μL)

SNV TagSet 2

Hybridization Buffer* 5

SNV Solid Tumor Probe T Pool working stock 0.5

SNV Solid Tumor Probe S Pool working stock 0.5

SNV Solid Tumor Probe M Pool working stock 2

Amplified DNA sample 5

Total Volume 15

*The Hybridization Buffer is included in the nCounter Master Kit, which must be purchased separately.

NOTE: The order of addition is important, so please follow the protocol exactly.

1. Pre-heat a thermal cycler to 65 ˚C with its heated lid turned on and the “forever” time setting.

2. Remove tubes of the SNV TagSet, SNV Solid Tumor Probe T Pool, SNV Solid Tumor Probe S Pool,

and SNV Solid Tumor Probe M Pool from the freezer and thaw at room temperature. Invert or

flick tube several times to mix well and briefly spin down reagents.

NOTE: Inspect the thawed tubes of SNV TagSet to make sure no colored precipitate is present. If

you see a colored precipitate, heat the entire tube to 75 ˚C for 10 minutes and cool at room

temperature before using. Do not heat the Probe S Pool above room temperature.



3. Create a working stock of Probe T by adding 22 μL of nuclease-free water or TE buffer directly to

the tube of SNV Solid Tumor Probe T Pool.

4. Create a working stock of Probe S by adding 22 μL of nuclease-free water or TE buffer directly to

the tube of SNV Solid Tumor Probe S Pool.

5. Create a working stock of Probe M by adding 22 μL of nuclease-free water or TE buffer directly to

the tube of SNV Solid Tumor Probe M Pool.

6. Create a hybridization master mix sufficient for 14 reactions, by adding the following directly to

the SNV TagSet tube that contains 28 μL, in the exact order as presented below:

70 μL of Hybridization Buffer

7 μL of SNV Solid Tumor Probe T working stock

7 μL of SNV Solid Tumor Probe S working stock

28 μL of SNV Solid Tumor Probe M working stock

Flick the tube repeatedly to mix then briefly spin down the hybridization master mix at less

than 1,000 RCF for less than 30 seconds.

7. Label the 12-strip PCR hybridization tubes supplied with the nCounter Master Kit. Ensure that the

tubes will fit in a microfuge or picofuge (cut the strip in half if necessary).

8. Add 10 μL of hybridization master mix from step 6 to each of the 12 tubes.

NOTE: Use a fresh tip for each pipetting step to accurately pipette the correct volume. The TagSet

has components that can wick up into the tip. You may not be able to dispense the correct amount

if you use the same tip to dispense master mix into all of the hybridization tubes.

IMPORTANT: Denaturation of prepared amplified DNA samples is critical for optimal

performance. The prepared DNA should have been recently denatured and chilled on ice during

steps 10 and 11 of DNA Sample Amplification. If the amplified DNA was stored frozen after

denaturation, repeat steps 10 and 11 from DNA Sample Amplification before continuing.

9. Add 5 μL of the denatured amplified DNA sample from DNA Sample Amplification to each

hybridization tube. Cap the tubes tightly and mix the reagents by flicking with a finger to ensure

complete mixing and then perform a final spin-down.

NOTE: Ensure that tube 12 receives 5 L of amplified DNA SNV Reference Sample (see the NOTE for Step 6 in DNA Sample Amplification).

10. Incubate DNA SNV hybridization reactions for at least 16 hours at 65 ˚C. Hybridization reactions

should be left at 65 ˚C until ready for processing. Maximum hybridization time should not

exceed 30 hours.

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11. Once the DNA SNV hybridization reactions have been removed from the thermal cycler,

proceed immediately to post-hybridization processing as described below in Lung SNV:Fusions

Post-Hybridization Processing. If storage is necessary, store DNA SNV hybridizations in a dark

place at room temperature for up to 6 hours. DNA SNV hybridization reactions may be stored

frozen (at -10 ° C or below) for up to one week prior to processing.



nCounter Vantage Lung Fusion Hybridization

GENERAL PROBE HANDLING INSTRUCTIONS:

During setup, do not vortex or pipette vigorously to mix as this may shear the Reporter Tags.

Mixing should be done by flicking or inverting the tubes.

If using a microfuge to spin down tubes, do not spin any faster than 1,000 RCF for more than 30 seconds.

Do not “pulse” to spin because that will cause the centrifuge to go to the maximum speed and may spin the TagSet out of solution.

To set up 12 reactions, a master mix is created for 14 reactions to allow for dead volume in pipetting.

IMPORTANT: Instructions for preparing the Probe A Pool, Probe B Pool, and Probe P Pool working stocks

are provided in steps 2–4 below. When making these working stocks, mix gently but thoroughly, such as

by flicking the tubes or performing several cycles of up-and-down pipetting to ensure obtaining

homogeneous working solutions.

Each hybridization reaction will contain the following components:

Table 10. Lung Fusion hybridization reaction components

Component Volume (μL)

nCounter XT TagSet 2

Hybridization Buffer* 5

Working Probe A Pool 0.5

Working Probe B Pool 0.5

Working Probe P Pool 0.5

RNA sample 6.5

Total Volume 15

*The Hybridization Buffer is provided with the nCounter Master Kit, which must be purchased separately.

1. Remove an aliquot of nCounter XT TagSet from the freezer and thaw it on ice. Invert several times to mix well, and briefly spin down the reagent at less than 1,000 RCF.

2. Create a 30X Working Probe A Pool by adding 22 μL of TE to the 3 μL Probe A Pool stock aliquot provided.

3. Create a 30X Working Probe B Pool by adding 22 μL of TE to the 3 μL Probe B Pool stock aliquot provided.

4. Create a 30X Working Probe P Pool by adding 22 μL of TE to the 3 μL Probe P Pool stock aliquot provided.

18


5. Each core TagSet tube contains 28 μL of reagent. Create a master mix by adding the following directly into the TagSet tube in the exact order presented:

70 μL of Hybridization Buffer

7 μL of the 30X Working Probe A Pool

7 μL of the 30X Working Probe B Pool

7 μL of the 30X Working Probe P Pool

6. Label a 12-tube strip.

7. Add 8.5 μL of the master mix from step 5 to each of the 12 tubes.

NOTE: Use a fresh tip for each pipetting step to accurately pipette the correct volume. The

TagSet has components that can wick up into the tip. You may not be able to dispense the

correct amount if you use the same tip to dispense master mix into all of the hybridization

tubes.

8. Add 6.5 μL of RNA sample from RNA Sample Preparation to each of the 12 tubes.

NOTE: Tube 12 should be reserved for control or reference RNA sample to run simultaneously with the DNA SNV Reference Sample (see the NOTE for Step 6 in DNA Sample Amplification). One may also choose to not use any RNA in lane 12. In this case, prepare a “blank” hybridization

reaction by adding 6.5 L of nuclease-free water or TE buffer.

9. If necessary, add nuclease-free water to each tube to bring the volume of each reaction to

15 μL.

10. Cap tubes and mix the reagents by inverting the tubes several times and flicking with a finger to ensure complete mixing. Briefly spin down and immediately place the tubes in the pre-heated 67° C thermal cycler with a heated lid set at 75° C.

11. Incubate reactions for at least 16 hours. Maximum hybridization time should not exceed 48 hours. Ramp reactions down to 4° C and process the following day. Do not leave the reactions at 4° C for more than 24 hours or increased background may result.

NOTE: The purpose of selecting a fixed hybridization time followed by a ramp down to 4° C is to ensure equivalent hybridization times of all assays being directly compared in the same series of experiments. Counts continue to accumulate with time, with total counts typically increasing 5% per hour between 16 and 24 hours. Although a 16-hour incubation is adequate for most purposes, a longer incubation will increase sensitivity by increasing counts without significantly increasing background.



Lung SNV:Fusions Post-Hybridization Processing

1. After the parallel hybridization reactions are completed, for each starting tissue or cell sample, pool the appropriate 15 μL of DNA Hybridization reaction from DNA SNV Hybridization and the appropriate (sample-matched) 15 μL of RNA Hybridization reaction from nCounter Vantage Lung Fusion Hybridization into a single sample tube within the nCounter 12-well strip tube. Each pooled sample will be processed and analyzed in a single nCounter cartridge lane.

2. For the lane 12 sample, if no control or reference Lung Fusion RNA hybridization reaction is to be used, substitute the “blank” hybridization reaction (see NOTE for Step 8 in nCounter Vantage Lung Fusion Hybridization).

3. Run the pooled 30 μL volume mixed hybridization reactions immediately on an nCounter Prep Station as described in the instrument manual.

20


Appendix: DNA SNV Reference Run

For accurate SNV detection, a reference data set is required during analysis of subsequently collected

experimental data for each system that is to be used (nCounter Prep Station, Digital Analyzer, and DNA

SNV Solid Tumor Panel lot). An extra SNV Panel kit will be provided with the order to be used as a

reference kit. Please do not use it for any other purposes. The components of the panel lot are listed in

Table 1: Materials and Reagents and the panel lot number that these components share is printed on

the DNA SNV Solid Tumor Panel box label. The panel lot does not include components provided in the

nCounter Master Kit.

It is important to keep a record of the specific Prep Station and Digital Analyzer (preferably by serial

numbers) and the DNA SNV Solid Tumor Panel lot number that were used to create a given reference

data set. Also send the information of the lot number from the DNA SNV Solid Tumor Panel box to the

[email protected] along with your instrument serial number.

To create a reference data set, follow the protocol as written, except in step 6 of DNA Sample

Amplification use the DNA SNV Reference Sample as the DNA for all 12 samples to be loaded into all 12

lanes of an nCounter cartridge. There is no need for additional DNA extraction.

During analysis of subsequently collected experimental sample data, the reference data set (all 12 lanes)

will be used. It is important to maintain a copy of this data set as RCC files for use within nSolver™

Analysis Software. Please also email this RCC file to [email protected]. This data will be used

later for data analysis.



Documents

nCounter Vantage 3D SNV:Fusions Lung Assay That Count® 3 NanoString Technologies® nCounter® Vantage 3D™ SNV:Fusions Lung Assay for MAX and FLEX Analysis Systems nCounter Vantage