PC-0247v2 QuantideX NGS Pan Cancer Kit Protocol

49612v1 – Effective Date: 2016-10

Protocol

For Research Use Only. Not for use in diagnostic procedures.

49559, 49560, 49561

48 - 192

49612

Asuragen, Inc. 2170 Woodward St. Austin, TX 78744-1840 USA +1.512.681.5200

Note: QuantideX® and QuantidexTM are considered to be synonymous labels/brands.

Asuragen® QuantideX® NGS Pan Cancer Kit Protocol

49612v1 – Effective Date: 2016-10 of 19

Table of Contents Protocol Overview ........................................................................................................................................................ 3

Intended Use ................................................................................................................................................................. 4

Test Principle ................................................................................................................................................................ 4

Kit Components ............................................................................................................................................................ 5

Kit Configuration ........................................................................................................................................................... 6

Required materials not provided .................................................................................................................................. 6

Optional equipment not provided ................................................................................................................................ 6

Limitations .................................................................................................................................................................... 7

Warnings and Precautions ............................................................................................................................................ 7

Pre-Analytical Steps ...................................................................................................................................................... 7

Library Prep Protocol .................................................................................................................................................... 8

Overview/Notes ........................................................................................................................................................ 8

DNA Assessment [Box 1] ........................................................................................................................................... 8

Gene-Specific PCR [Box 2] ......................................................................................................................................... 9

Tagging/Index PCR [Box 2 and Box 3] ..................................................................................................................... 10

Library Purification [Box 4] ..................................................................................................................................... 10

Library Quantification [Box 5] ................................................................................................................................. 11

Library Pooling/Normalization and Denature, Dilute, Load [Box 6] ....................................................................... 12

Generation of MiSeq Sample Sheet ............................................................................................................................ 13

Data Analysis ............................................................................................................................................................... 14

Expected Results of Kit Controls ................................................................................................................................. 14

Notice to Purchaser .................................................................................................................................................... 14

Symbol Legend ............................................................................................................................................................ 14

References .................................................................................................................................................................. 14

Appendix 1: Governing Equations .............................................................................................................................. 15

Appendix 2: Codon Regions Covered in the Pan Cancer Panel .................................................................................. 16

Appendix 3: Index Codes ............................................................................................................................................ 17

Related Protocols

PC-0235, QuantideX® DNA Assay, available at http://quantidex.asuragen.com

PC-0249, QuantideX® Reporter, available at http://quantidex.asuragen.com

MiSeq System User Guide, available at http://support.illumina.com/

Use these protocols in conjunction with the following protocol to successfully execute the QuantideX® Pan Cancer

Kit library prep workflow.

Contact Asuragen Technical Support if any problems are encountered when using this product:

[email protected]

+1.512.682.5200



QuantideX® NGS Pan Cancer Kit Protocol Overview

DNA Assessment

• Kit: QuantideX® DNA Assay [Box 1]

• 5 µL 2x QuantideX® Master Mix

• 0.5 µL each of Quant & Inhibition Primer Probe Mixes

• 0.05 µL ROX

• 2.95 µL Diluent

• 1 µL DNA Sample or DNA Standard (Plot samples against logarithmic standard curve to attain copy numbers)

Gene-Specific PCR

• Kit: QuantideX® Pan Cancer Panel [Box 2]

• 5 µL 2X Amplification Master Mix

• 1 µL Pan Cancer Primer Panel

• 4 µL DNA sample, or FFPE Positive Ctrl, or Multi-Variant Ctrl, or NTC

• (Suggested input range of 400 - 24000 functional copies)

Tagging PCR

• Kits: QuantideX® Pan Cancer Panel [Box 2] & QuantideX® Codes [Box 3]

• 7.5 µL 2X Index Master Mix

• 5.5 µL Index Code (AIL001-AIL192)

• 2 µL Gene-Specific PCR product

Library Purification

• Kit: QuantideX® Library Pure Prep [Box 4]

Library Quantification

• Kit: QuantideX® Library Quant [Box 5]

• 5 µL 2X LQ Master Mix

• 2 µL LQ Primer/Probe Mix

• 0.5 µL LQ ROX

• 0.5 µL LQ Standard

• 2 µL Purified product (diluted 10,000-fold in LQ Diluent) or LQ Positive Control

Pool, Denature,

Dilute, Load

• Kits: QuantideX® Sequencing Reagents [Box 6] & Illumina® MiSeq® Reagent Kit

• 15 µL 2.5 nM normalized library pool from stock purified products

• 3 µL 1 nM PhiX Control V3

• 2 µL 1 N NaOH; after adding NaOH, denature for 5 min at room temp, snap cool on ice

• Dilute 8 µL of the denatured library into 992 µL pre-chilled HT1 buffer

• Dilute 4 µL Read 1, Index Read, & Read 2 Primers in 636 µL HT1 buffer individually, load MiSeq®

qPCR using FAM & VIC channels

Temp Time Cycling

95°C 5 min -

95°C 15 sec 40 cycles

60°C 60 sec

Conc𝑙𝑖𝑏 = 25 𝑛𝑀 ∗ 2(𝛥𝐶𝑞𝑙𝑖𝑏 – 𝑍)

Temp Time Cycling

95°C 5 min -

95°C 30 sec

10 cycles 55°C 30 sec

72°C 60 sec

72°C 10 min -

4°C hold -

11 µL Library Pure Prep Beads

10 µL Tagging PCR product

Mix, pellet beads on magnet, remove supernatant

2 X 100 µL washes w/ EtOH-diluted Wash Buffer Leave plate on magnet

Elute in 20 µL Elution Buffer 1 min elute, recover pure products

Temp Time Cycling

95°C 5 min -


60°C 4 min


72°C 4 min

72°C 10 min -

4°C hold -

qPCR using FAM & VIC channels

Temp Time Cycling

95°C 5 min -


60°C 60 sec



Intended Use

The QuantideX® Pan Cancer Kit is intended for the quantification and enrichment of cancer-related variants in 46 regions of interest across 21 genes from DNA purified from human tissue or cell-lines. The kit supports multiplex next-generation sequencing analysis with an Illumina® MiSeq® instrument. The kit includes software (QuantideX® Reporter, P/N 49562) that analyzes MiSeq® data files for the identification of base substitution mutations and small insertions/deletions using a locally integrated bioinformatic pipeline and companion data visualization tools. See the Appendix for more details on the codons targeted by the QuantideX® Pan Cancer Kit.

Test Principle The kit includes reagents for DNA assessment, targeted enrichment, positive controls, index codes, and library purification and quantification, as well as an easy-to-use bioinformatics software solution. Both wet and dry bench processes are integrated within a simple workflow to enable analysis of low-quality and low-quantity DNA samples isolated from FFPE (Formalin-Fixed, Paraffin-Embedded) or FNA (Fine Needle Aspiration) tumor biopsies, fresh frozen tissue and cell lines.

Each kit component serves an essential role in the library prep workflow:

QuantideX® DNA Assay – Reagents for functional DNA quantification and sample assessment.

QuantideX® Pan Cancer Panel – PCR primers and PCR reagents that support single-well multiplex PCR enrichment across hotspot regions. Additionally, the box contains the following controls: o Pan Cancer Multi-Variant Ctrl – A pooled process control formulated from synthetic DNA in a background of cell line

genomic DNA designed to detect the most common variants in the QuantideX® Pan Cancer panel. o Pan Cancer FFPE Positive Ctrl – An authentic FFPE positive control sourced from tumor biopsies that presents a

common cancer mutation at or near the cutoff of 5% variant abundance, and is used at an input of 500 amplifiable copies.

QuantideX® Codes – Dual Index code oligonucleotide mixtures specific for the MiSeq and related Illumina sequencing platforms. These dual-index code primer mixes are formulated in racks of 48 codes with 4 uses per code (See Appendix 3).

QuantideX® Library Pure Prep – A proprietary magnetic bead chemistry that provides size selection and purification of the amplified libraries.

QuantideX® Library Quant – An assay that enables accurate assessment of purified libraries using a calibration-curve free method based on relative competitive quantitative PCR.

QuantideX® Sequencing Reagents – Custom sequencing primer mixtures for QuantideX NGS and standard Illumina library analysis.

QuantideX® Reporter – A push-button, integrated informatics processing and data reporting pipeline based on proprietary alignment and variant scoring algorithms.

Gene Codon Region Gene Codon Region

ABL1 249-258

HRAS

9-20

303-319 59-76

AKT1 16-27 113-121

AKT2 16-26 IDH1 122-134

ALK1 1174-1196

IDH2 138-145

1274-1278 163-174

BRAF 465-474 JAK2 607-620

591-612 KIT

557-579

EGFR

486-493 815-826

709-722

KRAS

4-15

737-761 55-65

767-798 104-118

849-861 137-148

ERBB2

755-769 MET 1245-1256

774-788

NRAS

9-20

839-847 55-67

877-883 110-119

FGFR1 123-136 144-150

250-262 PDGFRA

560-572

FGFR3

247-260 840-852

363-374 PIK3CA

540-551

638-653 1038-1049

FLT3 829-840 RET 916-926



QuantideX® NGS Pan Cancer Kit Components

Reference No. Description Cap color, if applicable

Vol/Rxns supported

Storage Temperature

49539 QuantideX® DNA Assay [Box 1] 100 Reactions -15 to -30°C

145339 Diluent clear 400 µL -15 to -30°C

145340-145343 DNA Standards (50, 10, 2, 0.4) white 16 µL each -15 to -30°C

145345 2x QuantideX® Master Mix yellow 500 µL -15 to -30°C

145336 Quant Primer Probe Mix blue 50 µL -15 to -30°C

145344 Inhibition Primer Probe Mix green 50 µL -15 to -30°C

145346 ROX amber 50 µL 2 to 8°C (after 1st use)

49550 QuantideX® Pan Cancer Panel [Box 2] 48 Reactions -15 to -30°C

145347 Pan Cancer Primer Panel violet 48 µL

-15 to -30°C

145348 2X Amplification Master Mix green 240 µL

145349 Pan Cancer FFPE Positive Ctrl clear 32 µL

145350 Pan Cancer Multi-Variant Ctrl white 32 µL

145361 2X Index Master Mix yellow 360 µL

49553-49556 QuantideX® Codes - Set A/B/C/D* [Box 3] 192 Reactions -15 to -30°C

150004-150007 Index Codes - Set A/B/C/D rack of codes 22 µL 2 to 8°C (after 1st use)

49551 QuantideX® Library Pure Prep [Box 4] 48 Reactions 2 to 8°C

145351 Library Pure Prep Beads amber 530 µL

2 to 8°C 145352 Wash Buffer bottle 2.5 mL

145353 Elution Buffer blue 1200 µL x 2

49552 QuantideX® Library Quant [Box 5] 48 Reactions -15 to -30°C

145354 LQ Diluent bottle 20 mL 2 to 8°C (after 1st use)

145355 LQ Positive Control green 16 µL -15 to -30°C

145356 LQ Standard white 24 µL -15 to -30°C

145357 LQ Primer/Probe Mix clear 96 µL -15 to -30°C

145358 2X LQ Master Mix yellow 240 µL -15 to -30°C

145359 LQ ROX amber 24 µL 2 to 8°C (after 1st use)

49557 QuantideX® Sequencing Reagents [Box 6] 8 Runs 2 to 8°C

145365 Sequencing Diluent blue 1200 µL

2 to 8°C 150001 Read 1 Sequencing Primers green 32 µLp

150002 Index Read Sequencing Primers green 32 µL

150003 Read 2 Sequencing Primers green 32 µL

*Only one of the four sets of Index Codes are included when ordering the QuantideX® Pan Cancer Kit. See Appendix for more details regarding the QuantideX® Codes.

Note: Box numbers are included on box labels.

Note: Reactions supported assumes approximately using 10% overage when preparing reaction master mixes.

The QuantideX® Reporter (49562), a bioinformatics solution for analyzing MiSeq output files, is included in the purchase of the kit. See the QuantideX® Reporter protocol, PC-0249, available at http://quantidex.asuragen.com for download.



Kit Configuration

Required materials not provided

Suggested part numbers have been provided for reagents and consumables, but equivalent products may be used.

Consult the MiSeq System User Guide for additional materials required to use Illumina MiSeq instrument.

Reagents Consumables Equipment MiSeq Reagent Kit

Nuclease-Free Water (Ambion AM9937)

Round-bottom plates (Evergreen 290-8117-01R)

qPCR instrument (ABI 7500 Series)†

Choose one of the following based on read depth needs‡

96-100% Ethanol (VWR 200004-484)

96-well PCR Plates (Phenix MPS-3580)

Thermal cycler (ABI 9700 or Veriti)

MiSeq Reagent Kit v3 (MS-102-3003)

1 N Sodium Hydroxide (VWR MK469360)

Adhesive PCR Film (AB-Gene AB-0558)

Magnetic plate stand (Ambion: AM10027)

MiSeq Reagent Kit v2 (MS-102-2002)

PhiX Control v3 (Illumina 15017666)

Optical 96-well plate (ABI 4306737)

Illumina MiSeq Desktop Sequencer

MiSeq Reagent Kit v2 Micro (MS-103-1002)

96-100% Isopropanol (VWR MK304306)

Optical adhesive film (ABI 4311971)

Micronic tube decapper (Univo SR008, or equiv.)

MiSeq Reagent Kit v2 Nano (MS-103-1001)

† If an ABI 7500 instrument is unavailable for LQ PCR, consult Asuragen Tech Support for guidance on use of other platforms.

‡ Consult Illumina or Asuragen Tech Support for assistance.

Optional equipment not provided

Flatbed or handheld scanner with Data Matrix 2D barcode reading capabilities



Limitations

Sample Input

The concentration of the DNA sample must be determined as functional or amplifiable copies per microliter (cp/uL), and annotated with each sample for sequence analysis and variant calling.

o Use the QuantideX® DNA Assay to obtain copy number concentrations for each sample. o Samples with less than a 100 cp/uL may be analyzed, but are at-risk for inaccuracies due to the

reduced template diversity available within that sample for amplification and variant detection. o For best results, load ≥ 400 amplifiable copies into the Gene-Specific PCR enrichment. o Samples with greater than 6000 cp/µL should be diluted 10-fold in nuclease free water prior to

library prep.

Rare Single Nucleotide Polymorphisms (SNPs)

The QuantideX® library preparation method is based on PCR amplification of targeted regions of cancer-associated genes (see Appendix). The presence of rare SNPs in the primer binding region may result in reduced amplicon yield or allele dropout.

qPCR Instrument

The LQ PCR protocol has been verified for use on the ABI 7500 platform in standard mode. Analysis in Fast mode or use of other real-time instruments is not supported at this time.

Contact Asuragen Tech Support for guidance on performing qPCR on an instrument other than ABI 7500.

Reagent Stability

The reagents should be used within their labeled expiry and stored according to table above.

Reagents stored frozen (-15 to -30°C) are formulated to support eight (8) freeze-thaw cycles.

MiSeq Instrument Control

The PhiX sequencing control must be included in each NGS run for cluster diversification and to aid in troubleshooting.

Instrument/Platform compatibility

This protocol was written to support MiSeq instrument analysis. Specific modifications may be required for other Illumina Sequencing platforms (e.g. the NextSeq system).

A separate protocol guide may be used to describe use of these reagents for alternative platforms

Warnings and Precautions

Use proper PPE. Wear appropriate protective eyeglasses, protective gloves, and protective clothing when working with these materials.

Follow Universal Precautions in compliance with OSHA 1910:1030, CLSI M29, or other applicable guidance when handling human samples.

Use nuclease-free filter pipette tips and nuclease-free tubes.

PCR carry-over contamination can result in false-positive signals. Use appropriate precautions in sample handling, workflow, and pipetting.

Do not pool components from different reagent lots.

Prior to use, ensure that all instruments are calibrated according to the manufacturer’s instructions.

Pre-Analytical Steps

It is not necessary to obtain amplifiable copy numbers for either of the controls included in the kit. They are provided at known concentrations.

o Pan Cancer FFPE Positive Ctrl (145349): 125 cp/µL o Pan Cancer FFPE Multi-Variant Ctrl (145350): 750 cp/µL



QuantideX® NGS Pan Cancer Kit Library Prep Protocol

Overview/Notes

Track sample ID, amplifiable copy number, and assigned index code carefully throughout the procedure. All three are critical for successful sequencing analysis.

Use this protocol in conjunction with Illumina’s MiSeq System User Guide.

Unless noted, all master mixes and reactions may be prepared at room temperature.

When combining reagents as instructed, pipette up and down 3-5 times to ensure even dispensing.

Replicate testing in DNA Assessment and Library Quant (LQ) qPCR assays is not required.

Library prep can be safely stopped after each major reaction in the procedure. o After the reaction is complete, store sample plate(s) at +2 to +8°C for short term (≤ 24 hrs) or -15

to -30°C otherwise. Keep all reaction plates until sequencing data has been obtained. o When ready to proceed, bring plate to room temperature with other required reagents.

Prior to each reaction, o Bring each required reagent and/or plate to room temperature for at least 15 minutes. o Briefly vortex and centrifuge each component and/or plate prior to opening.

Exception: Do not centrifuge the Library Pure Prep Beads.

DNA QC [Box 1]

Reference the standalone protocol, PC-0235: QuantideX® DNA Assay Protocol, as needed for using Box 1.

1. Prepare a QC PCR master mix in a clean microcentrifuge tube. A. Add the reagents to the tube in the order listed; volumes are shown per reaction.

Description Vol. (µL)

2x QuantideX® Master Mix 5.0

Quant Primer Probe Mix 0.5

Inhibition Primer Probe Mix 0.5

Diluent 2.95

ROX 0.05

Total master mix per well 9.0

Note: If the volume of ROX to add is too small to pipet, dilute an aliquot 5-fold in Diluent, then add 5X the amount of diluted ROX. With this option, add proportionately less Diluent to the master mix.

B. Prepare sufficient master mix for the total number of samples tested, including the 4 DNA standards in duplicate and an NTC sample (# of samples + 9).

C. Mix the master mix with gentle flicking/vortexing, then pulse spin to collect contents.

2. Aliquot 9 µL of QC PCR master mix to separate wells in a 96-well optical PCR plate.

3. Add 1 µL of each DNA Standard to separate wells in duplicate (8 wells total).

4. Add 1 µL of Diluent to NTC well.

5. Add 1 µL of each DNA sample to separate wells.

6. Seal plate with optical seal, vortex to mix, centrifuge 1 min at 1500 g.

7. Perform the 10 µL qPCR reaction on an ABI 7500 with the following parameters:

o FAM and VIC detectors (both applied to every well, no quenchers) o ROX Passive Reference o Cycling conditions as shown, standard mode o Collect data during 60°C step. o Auto Baseline; Manual Threshold: 0.1

DNA QCGene-Specific

PCRTagging PCR



Pool, Denature, Dilute, Load

Important: Obtaining and tracking

the amplifiable copy number for

each DNA sample is critical not only

for library prep input, but also for

downstream analysis with

QuantideX® Reporter.

Example of a 7-sample plate layout:

1 2 3

A Std (50) Sample1

B Std (50) Sample2

C Std (10) Sample3

D Std (10) Sample4

E Std (2) Sample5

F Std (2) Sample6

G Std (0.4) Sample7

H Std (0.4) NTC

Temp Time Cycling

95°C 5 min -


60°C 60 sec



8. Analysis

A. Determine Copy Input

Plot a logarithmic standard curve of the DNA Standards, using copy number and FAM Cq values. Plot the FAM Cq values from DNA samples on the curve, calculate copy numbers for samples.

Record all sample copy numbers for analysis by the QuantideX® Reporter.

B. Determine Inhibition Status

Sample VIC Cq < (2 + Avg VIC Cq of Standard Curve), sample is considered uninhibited Sample VIC Cq ≥ (2 + Avg VIC Cq of Standard Curve), indicates presence of PCR inhibitors

C. If needed, data can be uploaded and interpreted at http://quantidex.asuragen.com/ to attain copy number and inhibition data on DNA samples.

D. Input recommendations into QuantideX® Pan Cancer library prep:

≤ 100 cp/µL: may proceed to QuantideX® Pan Cancer library prep, but at-risk for call inaccuracies due to potentially limiting functional template quantities 100 - 6,000 cp/µL: recommended input for QuantideX® Pan Cancer library prep > 6,000 cp/µL: dilute an aliquot of the sample 10-fold in nuclease-free water for use in

QuantideX® Pan Cancer library prep (e.g. 2 µL sample + 18 µL water).

Gene-Specific PCR [Box 2]

1. Prepare a GS PCR master mix in a clean microcentrifuge tube. A. Add the reagents to the tube in the order listed;

volumes are shown per reaction.

B. Create enough master mix for samples of interest plus the 3 controls noted in step 4.

C. Gently vortex to mix, and briefly centrifuge the GS PCR master mix.

2. Aliquot 6 µL of the GS PCR master mix into separate wells of a clean 96-well plate.

3. Add 4 µL of each DNA sample of interest to separate wells containing GS PCR master mix.

Note: Recommended DNA input range is 400 - 24,000 copies (100 cp/µL - 6,000 cp/µL)

4. Add 4 µL of each control to separate wells containing GS PCR master mix:

A. Pan Cancer FFPE Positive Ctrl B. Pan Cancer Multi-Variant Ctrl C. Nuclease-free water (NTC)

5. Seal the plate, gently vortex to mix, briefly centrifuge the plate to collect contents.

6. Perform the 10 µL GS PCR reaction on a thermal cycler with the following conditions:

DNA QCGene-Specific

PCRTagging PCR





2X Amplification Master Mix 5

Pan Cancer Primer Panel 1

Total master mix per well 6

Description Conc. (cp/µL)

DNA Standard (50) 15150



DNA Standard (0.4) 121

Step Temp. Time Cycling

Enzyme Activation 95°C 5 min -

Denature 95°C 15 sec 2 cycles

Anneal, Extend 60°C 4 min

Denature 95°C 15 sec 23 cycles

Anneal, Extend 72°C 4 min

Final Extension 72°C 10 min -

End Reaction, Hold 4°C hold -

Including the provided controls in each library prep batch helps to ensure run validity, monitor operator proficiency, and track performance trends over time.

- Pan Cancer FFPE Positive Ctrl: FFPE DNA at or near the cutoff of 5% variant and 500 copy input.

- Pan Cancer Multi-Variant Ctrl: pooled synthetic DNA control in a background of cell line genomic DNA designed to detect the most common variants.

- NTC (water): will reveal any contamination in the workflow



Tagging/Index PCR [Box 2 and Box 3]

1. Extra Setup A. Centrifuge the rack of index codes for 1 minute at 2000 x g prior to each use. B. If needed, scan rack of codes with a barcode scanner to ensure correct positions of index codes.

Refer to the provided COA as needed. C. Assign each sample/control from GS PCR a unique Index Code (AIL001-AIL192), and record this

information by plate location and sample ID.

Critical: Each sample must be assigned (and receive) a different index code.

2. In a new 96-well plate, for each GS PCR product: A. Aliquot 7.5 µL 2X Index Master Mix B. Add 5.5 µL Index Code (AIL###, unique to each well)

3. Transfer 2 µL GS PCR product to corresponding wells of Tag PCR plate.

Important: Ensure the correct Index Code was added to the appropriate well according to the previously-made assignments, and that each well received a different Index Code.

4. Seal the Tag PCR plate, gently vortex to mix, and briefly centrifuge the plate to collect contents. 5. Perform the 15 µL Tagging PCR reaction on a

thermal cycler with the following conditions.

Library Purification [Box 4]

1. Extra Setup A. If this is the first use of the kit, add 10 mL of 100% Ethanol to Wash Buffer bottle, cap the bottle,

and mix well by inverting the bottle several times. 10 mL of 100% ethanol must be added to the Wash Buffer bottle. Failure to add ethanol to the Wash Buffer will result in sample and reagent loss.

B. Fully resuspend Library Pure Prep Beads with gentle vortexing for 30 - 45 seconds, pulsing every 5 - 10 seconds.

DO NOT centrifuge the Library Pure Prep Beads. C. Ensure reagents and consumables are readily available before starting the purification. It is

important to not let the beads dry out until the very end of the procedure. 2. Bind the Tag PCR product to the magnetic beads

A. Aliquot 11 µL Library Pure Beads to each well in a clear, round-bottom, 96-well plate (Evergreen Scientific: 290-8117-01R). Note: To keep bead concentration consistent across the plate while aliquotting, cap the Library Pure Prep Beads every 4 wells and gently pulse vortex.

B. Add 10 µL Tag PCR products to the wells of beads, mix by pipetting until mixture appears homogenous. 5 µL Tag PCR product will remain in Tag PCR plate. Note: Avoid creating bubbles while mixing.

DNA QCGene-Specific

PCRTagging PCR




DNA QCGene-Specific

PCRTagging PCR






Denature 95°C 30 sec 10

cycles Anneal 55°C 30 sec

Extend 72°C 60 sec

Final Extension 72°C 10 min -

End Reaction, Hold 4°C hold -



3. Wash the bead-bound libraries: A. Place plate on magnetic stand (Ambion: AM10027) to pellet beads out of solution (≈15 seconds). B. Leaving the plate on the magnetic stand, slowly remove and discard the clear supernatant (≈20µL)

from each well, taking care to avoid the bead pellet. C. Leaving the plate on the magnetic stand, add 100 µL of Wash Buffer containing EtOH to each well. D. Wait for beads to re-pellet if they were disrupted when adding the Wash Buffer (≈15 seconds). E. Leaving the plate on the magnetic stand, slowly remove and discard the clear supernatant from

each well, taking care to avoid the bead pellet. F. Repeat the previous necessary steps to perform another wash for a total of two washes, removing

as much buffer as possible after the second wash. 4. Elute the purified libraries:

A. After removal of all Wash Buffer, incubate plate on the magnet at room temperature for 1 minute to dry the beads. Note: Excessive drying of beads (>2 min) may lead to loss of product.

B. Remove the plate from the magnet. C. Add 20 µL of Elution Buffer to each well, and mix by pipetting until bead pellets are fully

resuspended and elution reaction appears homogenous. Use pipette tip in excess of 20 µL.

Notes: Beads may clump or appear granular in solution during this step. This is acceptable, and does not affect the elution.

D. Incubate plate for 1 minute on bench top once all bead pellets are resuspended. i. 1 minute is a minimum incubation time; samples with low GS PCR copy input (<400 cp)

can elute for up to 4 minutes. E. Place plate on magnet to pellet beads out of solution (≈15 seconds). F. Transfer 18 µL of the clear supernatant into a clean 96-well plate, taking care to avoid the beads. G. This resultant plate contains the purified libraries. An aliquot of the purified libraries will be

quantified (see next step) prior to pooling.

Library Quantification [Box 5]

1. Dilute an aliquot of purified library 10,000-fold (via two 100-fold serial dilutions) in LQ Diluent using the following procedure. (Note: Both dilutions are discarded after the products have been quantified.)

A. Transfer 198 µL LQ Diluent to each well in a 96-well plate. B. Add 2 µL of purified library, pipette up and down 10 times to ensure a full dispense. C. Mix by pipetting 10 times with a pipet set to 150 µL. D. Add 198 µL LQ Diluent to clean wells in a 96-well plate. E. Transfer 2 µL of 1:100 diluted library, pipette up and down 10 times to ensure a full dispense. F. Mix by pipetting 10 times with a pipet set to 150 µL. G. After first use, store LQ Diluent at 2 to 8°C.

Warning: The LQ Diluent is prone to bubbles. Avoid aspirating air while performing these dilutions.

2. Prepare an LQ PCR master mix in a clean microcentrifuge tube.

A. Add the reagents to the tube in the order listed; volumes are shown per reaction.


2X LQ Master Mix 5

LQ Primer/Probe Mix 2

LQ ROX 0.5

LQ Standard 0.5

Total master mix per well 8.0

B. Create enough master mix for all diluted purified products plus 1 control.

DNA QCGene-Specific

PCRTagging PCR






C. Gently vortex the tube to mix, and briefly centrifuge the LQ PCR master mix. D. After first use, store LQ ROX at 2 to 8°C.

3. Aliquot 8 µL LQ PCR master mix into a 96-well optical plate, including one extra well for a control.

4. Add 2 µL of 10,000-fold diluted purified products.

5. Add 2 µL LQ Positive Control (undiluted, neat from tube) to the extra well.

5. Seal the plate, gently vortex to mix, and briefly centrifuge the plate to collect contents.

6. In SDS software, assign FAM and VIC detectors for each sample in order to detect purified library and competitive standard (LQ Standard), respectively.

7. Follow ABI 7500 protocol to perform LQ PCR with the following conditions/parameters in standard mode:

- Collect data during 60°C step - Automatic Baseline; Manual Threshold at 0.1 - FAM & VIC detectors (no quenchers)

8. Calculate the concentration (in nM) of each stock purified sample library with the following formula:

𝐶𝑜𝑛𝑐𝑙𝑖𝑏 = 25 nM ∗ 2(∆𝐶𝑞𝑙𝑖𝑏−𝑍) (Note: See Appendix for derivation of LQ PCR formula.)

ΔCqlib = CqVIC – CqFAM of the library;

Z = ΔCqPosCtrl = CqVIC – CqFAM for the LQ Positive Control.

Discard the dilution plates before proceeding to the next step.

Library Pooling/Normalization and Denature, Dilute, Load [Box 6]

Use the following instructions in conjunction with Illumina’s instructions for loading and running the MiSeq.

1. Setup

A. Follow the manufacturer’s (Illumina) instructions for thawing and preparing the MiSeq Reagent cartridge for loading.

B. If needed, dilute stock PhiX control to 1 nM in Sequencing Diluent. C. If needed, dilute stock NaOH to 1 N in nuclease-free water.

2. Pooling/normalizing the libraries based on their respective concentrations from LQ PCR:

A. Determine the median concentration of all the prepared libraries, Concmedian. B. Use the formula below to determine how much volume of each individual sample library to add to

the pool. The formula targets 5 µL per library.

𝑉𝑜𝑙𝑙𝑖𝑏 = 𝐶𝑜𝑛𝑐𝑚𝑒𝑑𝑖𝑎𝑛

𝐶𝑜𝑛𝑐𝑙𝑖𝑏 ∗ 5 µ𝐿

For ease of pipetting, round the calculated volume to the nearest microliter.

C. Transfer the calculated, rounded volume of each library to a clean microcentrifuge tube. D. Do not pipet less than 2 µL of any one library, and no more than 15 µL. E. For NTCs, transfer only 4 µL of purified product, regardless of the calculated concentration.

3. Using the rounded volumes used of each library, calculate the total concentration of the pool:

𝐶𝑜𝑛𝑐𝑝𝑜𝑜𝑙 = (𝐶𝑜𝑛𝑐𝑙𝑖𝑏 ∗ 𝑉𝑜𝑙𝑙𝑖𝑏)1 + (𝐶𝑜𝑛𝑐𝑙𝑖𝑏 ∗ 𝑉𝑜𝑙𝑙𝑖𝑏)𝑖

𝑉𝑜𝑙𝑝𝑜𝑜𝑙 where “i” is each library

4. Dilute the library pool to 2.5 nM in Sequencing Diluent in a clean microcentrifuge tube.

DNA QCGene-Specific

PCRTagging PCR






Denature 95°C 15 sec 40 cycles Anneal/Extend 60°C 60 sec



5. Transfer 992 µL of the HT1-hyb buffer to a clean microcentrifuge tube, place on ice.

6. Denature the pooled library: A. Transfer 15 µL of the 2.5 nM pool to a clean microcentrifuge tube. B. Add 3 µL of 1 nM PhiX control C. Add 2 µL of 1 N NaOH, immediately cap the tube, vortex to mix, briefly centrifuge. D. Incubate the tube for 5 minutes at room temperature. E. Place tube on ice for at least 2 minutes. This tube now holds the denatured library.

7. Dilute the denatured library mixture: A. Transfer 8 µL of the denatured library to the pre-chilled 992 µL aliquot of HT1-hyb buffer. (This

1000 µL is the sample to be loaded into the MiSeq cartridge in position 17.) B. Vortex to mix, briefly centrifuge to collect contents, place back on ice.

8. Dilute the sequencing primers for loading onto the MiSeq. In 3 separate microcentrifuge tubes: A. Add 4 µL Read 1 Sequencing Primers to 636 µL HT1-hyb buffer. B. Add 4 µL Index Read Sequencing Primers to 636 µL HT1-hyb buffer. C. Add 4 µL Read 2 Sequencing Primers to 636 µL HT1-hyb buffer. D. Vortex all to mix, briefly centrifuge, place on ice until ready to load into MiSeq cartridge.

9. Load the MiSeq cartridge: A. Add 600 µL of the pooled, diluted, denatured library to position 17. B. Add 600 µL diluted Read 1 Sequencing Primers to position 18. C. Add 600 µL diluted Index Read Sequencing Primers to position 19. D. Add 600 µL diluted Read 2 Sequencing Primers to position 20.

Follow manufacturer’s (Illumina) instructions on loading and running the MiSeq instrument.

Generation of MiSeq Sample Sheet

1. Download Illumina’s IEM software at Illumina.com. 2. Add the “ASGN Quantidex Pan Cancer DNA Panel.txt” protocol file into the appropriate location to

integrate with the IEM software (e.g.: C:\Program Files\Illumina\Illumina Experiment Manager\SamplePrepKits).

3. Replace the “Generate FASTQ.txt” file found in <C:\Program Files\Illumina\Illumina Experiment Manager\Applications> with the Asuragen-provided version of the .txt file. Note: Both of the .txt files mentioned above are included with the purchase of QuantideX® Pan Cancer products, and also available at quantidex.asuragen.com.

4. Follow Illumina’s instructions to generate the sample sheet in the IEM software, adhering to the following parameters: Sample Prep Kit Type: ASGN Quantidex Pan Cancer DNA Panel Index Reads: 2 Assign Index Codes (AIL001-192) I-5 and I-7 to correct sample IDs, making sure they match the

assignments made during Tagging/Index PCR. Refer to provided QuantideX® Codes COA as needed. Instrument: MiSeq Application Category: Other Application: FASTQ only FASTQ only run settings:

Cycles Read 1: 150

Cycles Read 2: 150 FASTQ only Workflow-Specific settings:

Custom Primer for Read 1

Custom Primer for Index

Custom Primer for Read 2 5. Transfer the resulting .csv file to the MiSeq instrument, follow Illumina’s guide on loading and running the

MiSeq.



Data Analysis

A completely integrated bioinformatics suite, the QuantideX® Reporter software (49562), is available with

purchase of the kit at http://quantidex.asuragen.com, along with its protocol (PC-0249). The patent-pending

software requires the copy input for each sample determined from the DNA Assay to inform the variant caller.

Expected Results of Kit Controls

DNA Assay Standards: coefficient of determination (r2) > 0.96

LQ Standard: 15 < Cq < 20

Pan Cancer FFPE Positive Ctrl: mutant BRAF V600E detected between 3.5 – 10.0%

Pan Cancer Multi-Variant Ctrl: 14 variants detected between 3.0 – 30.0% variant

o BRAF_p.V600E o KIT_p.D816V

o EGFR_p.E746_A750delELREA o KRAS_p.G12D

o EGFR_p.L858R o KRAS_p.Q61R

o EGFR_p.T790M o NRAS_p.G12D

o EGFR_p.V769_D770insASV o NRAS_p.Q61R

o HRAS_p.G12D o PIK3CA_p.E545K

o HRAS_p.Q61R o PIK3CA_p.H1047R

Notice to Purchaser

This product is intended for research use only. It is not intended for diagnostic use.

This product may not be resold, modified for resale, or used to manufacture commercial products without the written approval of Asuragen.

QuantideX® and QuantidexTM are trademarks of Asuragen, Inc and considered synonymous labels/brands.

All other names, logos, and other trademarks are the property of their respective owners.

The QuantideX® Library Quant Kit was optimized with ABI 7500 real-time instruments. No other instrument is supported at this time. Contact Asuragen Tech Support for alternative quantification methods.

Information in this document is subject to change. Asuragen assumes no responsibility for any errors that may appear in this document. In no event shall Asuragen be liable in any way (whether in contract, tort (including negligence) or otherwise) for any claim arising in connection with or from the use of this product. Nothing in this document excludes or limits any liability which it is illegal for Asuragen to exclude or limit.

Symbol Legend

Symbol Description

Catalog number

Batch code

Contains sufficient reagents for N reactions

Consult instructions before use

Temperature limitation

Use by

Manufactured by

References

Hadd, A.G., et al., J Mol Diagn, 2013. 15(2): p. 234-47.

Sah et al. Genome Medicine 2013, 5:77



Appendix 1: Governing Equations

Example governing equations are included to show derivation of key terms and to support incorporation of protocol steps into separate workflows or analysis tools.

Calculate concentration of purified library via Library Quant (LQ) PCR data The concentration of each sample is determined using a comparative qPCR (cqPCR) method. The difference in Ct values from the FAM channel (representing the NGS library) to the VIC channel (representing the competing LQ Standard of a known concentration) is the basis for quantifying the purified library. A formula is used to quantify each library. Fundamentally, this equation is:

𝐶𝑜𝑛𝑐𝑙𝑖𝑏 = 𝐶𝑜𝑛𝑐𝑠𝑡𝑑 ∗ 𝑒∆𝐶𝑞 Conclib = concentration of the library sample Concstd = concentration of the standard, known to be 10 pM e = PCR amplification efficiency, which is equivalent to 2 ΔCqlib = the observed difference in Cq values between the VIC and the FAM channels (CqVIC - CqFAM) for the library For the purpose of this workflow, additional terms need to be accounted for, such as the difference in volume between sample and standard added to the PCR reaction, how much the sample is diluted before PCR, and a run-specific correction factor related to the LQ Positive Control. Taking these into account, a more encompassing formula is used:

𝐶𝑜𝑛𝑐𝑙𝑖𝑏 ∗ 𝑉𝑜𝑙𝑙𝑖𝑏 = 𝐶𝑜𝑛𝑐𝑠𝑡𝑑 ∗ 𝑉𝑜𝑙𝑠𝑡𝑑 ∗ 𝑒∆𝐶𝑞𝑙𝑖𝑏−𝑍 ∗ 𝐷𝐹 Vollib = volume of library sample in the PCR reaction, known to be 2 µL Volstd = volume of standard in the PCR reaction, known to be 0.5 µL Z = ΔCqPosCtrl = CqVIC – CqFAM of LQ Positive Control (this assumes use of ABI 7500 platform) DF = Dilution Factor of the library, known to be 10,000 Dividing by Vollib can isolate the Conclib term. And lastly, while the LQ Standard concentration is known to be in pM (pmol/L), library concentration is most appropriately reported in nM (nmol/L). This requires a unit conversion, and completes the formula for determining purified library concentration using QuantideX® Library Quant:

𝐶𝑜𝑛𝑐𝑙𝑖𝑏 = 𝐶𝑜𝑛𝑐𝑠𝑡𝑑 ∗𝑉𝑜𝑙𝑠𝑡𝑑

𝑉𝑜𝑙𝑙𝑖𝑏∗ 𝑒∆𝐶𝑞𝑙𝑖𝑏−𝑍 ∗ 𝐷𝐹 ∗

1 𝑛𝑀

1000 𝑝𝑀

Plugging in all the known values:

𝐶𝑜𝑛𝑐𝑙𝑖𝑏 = 10 𝑝𝑀 ∗0.5 𝑢𝐿

2 𝑢𝐿∗ 2∆𝐶𝑞𝑙𝑖𝑏−𝑍 ∗ 10000 ∗

1 𝑛𝑀

1000 𝑝𝑀

…and then reducing:

𝐶𝑜𝑛𝑐𝑙𝑖𝑏 = 25 𝑛𝑀 ∗ 2(∆𝐶𝑞𝑙𝑖𝑏−𝑍)



Appendix 2: Codon Regions Covered in the Pan Cancer Panel

Gene Codon Region Chromosome ROI Start ROI End

ABL1 249-258 9 133738340 133738376 303-319 9 133748247 133748295

AKT1 16-27 14 105246517 105246553

AKT2 16-26 19 40762928 40762961

ALK 1174-1196 2 29432652 29432675 1274-1278 2 29443631 29443699

BRAF 465-474 7 140453097 140453166 591-612 7 140481385 140481415

EGFR 486-493 7 55227989 55228012 709-722 7 55241677 55241717 737-761 7 55242439 55242513 767-798 7 55249001 55249096 849-861 7 55259487 55259525

ERBB2 755-769 17 37880218 37880263 774-788 17 37880990 37881032 839-847 17 37881323 37881351 877-883 17 37881436 37881457

FGFR1 123-136 8 38282175 38282215 250-262 8 38285902 38285947

FGFR3 247-260 4 1803562 1803604 363-374 4 1806065 1806105 638-653 4 1807852 1807900

FLT3 829-840 13 28592623 28592661

HRAS 9-20 11 533538 533567 59-76 11 533826 533881 113-121 11 534261 534300

IDH1 122-134 2 209113104 209113143

IDH2 138-145 15 90631830 90631866 163-174 15 90631918 90631941

JAK2 607-620 9 5073740 5073776

KIT 557-579 4 55593603 55593671 815-826 4 55599315 55599352

KRAS 4-15 12 25398273 25398311 55-65 12 25378644 25378689 104-118 12 25380262 25380297 137-148 12 25398273 25398311

MET 1245-1256 7 116423403 116423441

NRAS 9-20 1 115252190 115252211 55-67 1 115252282 115252314 110-119 1 115256509 115256548 144-150 1 115258720 115258759

PDGFRA 560-572 4 55141032 55141071 840-852 4 55152086 55152124

PIK3CA 540-551 3 178936075 178936112 1038-1049 3 178952055 178952092

RET 916-926 10 43617407 43617443



Appendix 3: Index Codes

QuantideX® Codes - Set A 49553 ; 150004

QuantideX® Codes - Set B 49554 ; 150005

QuantideX® Codes - Set C 49555 ; 150006

QuantideX® Codes - Set D 49556 ; 150007

Set ID I7 Index

ID I5 Index

ID Set ID

I7 Index ID

I5 Index ID

Set ID I7 Index

ID I5 Index

ID Set ID

I7 Index ID

I5 Index ID

AIL001 I7-001 I5-01 AIL049 I7-007 I5-01 AIL097 I7-013 I5-01 AIL145 I7-019 I5-01
















































Index region sequences of the Index Codes can be found in the ASGN QuantideX Pan Cancer DNA Panel.txt file distributed with the purchase of the kit, or available at http://quantidex.asuragen.com.



QuantideX® Codes Rack Layouts

QuantideX® Codes - Set A (49553; 150004)

I7 Index ID I7-001 I7-002 I7-003 I7-004 I7-005 I7-006

I5 Index ID 1 2 3 4 5 6 7 8 9 10 11 12

I5-01 A AIL001 AIL009 AIL017 AIL025 AIL033 AIL041 AIL049 AIL057 AIL065 AIL073 AIL081 AIL089

I5-02 B AIL002 AIL010 AIL018 AIL026 AIL034 AIL042 AIL050 AIL058 AIL066 AIL074 AIL082 AIL090

I5-03 C AIL003 AIL011 AIL019 AIL027 AIL035 AIL043 AIL051 AIL059 AIL067 AIL075 AIL083 AIL091

I5-04 D AIL004 AIL012 AIL020 AIL028 AIL036 AIL044 AIL052 AIL060 AIL068 AIL076 AIL084 AIL092

I5-05 E AIL005 AIL013 AIL021 AIL029 AIL037 AIL045 AIL053 AIL061 AIL069 AIL077 AIL085 AIL093

I5-06 F AIL006 AIL014 AIL022 AIL030 AIL038 AIL046 AIL054 AIL062 AIL070 AIL078 AIL086 AIL094

I5-07 G AIL007 AIL015 AIL023 AIL031 AIL039 AIL047 AIL055 AIL063 AIL071 AIL079 AIL087 AIL095

I5-08 H AIL008 AIL016 AIL024 AIL032 AIL040 AIL048 AIL056 AIL064 AIL072 AIL080 AIL088 AIL096

QuantideX® Codes - Set B (49554; 150005)

I7 Index ID I7-007 I7-008 I7-009 I7-010 I7-011 I7-012

I5 Index ID 1 2 3 4 5 6 7 8 9 10 11 12









QuantideX® Codes - Set C (49555; 150006)

I7 Index ID I7-013 I7-014 I7-015 I7-016 I7-017 I7-018

I5 Index ID 1 2 3 4 5 6 7 8 9 10 11 12









QuantideX® Codes - Set D (49556; 150007)

I7 Index ID I7-019 I7-020 I7-021 I7-022 I7-023 I7-024

I5 Index ID 1 2 3 4 5 6 7 8 9 10 11 12









The codes are based on separate dual index code combinations and are interchangeable for analysis.

PC-0247v2 QuantideX NGS Pan Cancer Kit Protocol

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Transcript of PC-0247v2 QuantideX NGS Pan Cancer Kit Protocol