The QuantStudio® 3D Digital PCR Chip contains 20,000 individual PCR reaction wells and works by partitioning a standard PCR reaction into thousands of individual PCR reactions. For highly concentrated samples such as a typical NGS library, upfront sample dilution helps ensure that a portion of these partitions contain the target molecule, while other partitions do not, leading to positive and negative reactions. Following amplification on a dual flat block thermal cycler, the fraction of negative reactions is used to quantify the number of target molecules in the sample, all without reference to standards or controls. This application note describes a simple workflow for quantifying Illumina® NGS libraries using the QuantStudio® 3D Digital PCR System.

Library quantification in the Illumina® NGS workflowA high-level NGS workflow is illustrated in Figure 1. In summary, once a library has been constructed, the QuantStudio® 3D Digital PCR System is used to directly measure the number of molecules that contain both library adapters and can be sequenced. Based on this measurement, the appropriate library concentration is used to achieve optimal cluster density. Following clonal amplification, sequencing is performed on an Illumina® MiSeq®, HiSeq®, or NextSeq™ 500 platform.

IntroductionIllumina® MiSeq®, HiSeq®, and NextSeq™ 500 platforms utilize unordered solid-phase amplification of library clones to generate clonal sequence clusters (bridge PCR) prior to next-generation sequencing (NGS). This cluster generation step is critical to obtaining maximal sequence data quality and yields, and is highly dependent on the library input amount. Current detection methods for quantifying NGS libraries (i.e., Fragment Analyzer™, Bioanalyzer®, and spectrophotometer readings) are not able to specifically measure only those fragments that have incorporated both library adapters. This commonly results in a miscalculation of library concentration, leading to reduced throughput. As a result, an accurate and precise method for quantifying libraries prior to cluster generation is critical to maximizing throughput from a sequencing run.

Real-time PCR is an effective approach for NGS library quantification, but it relies on a comparison to a standard curve for absolute concentration measurement. Generation of the standard curve requires a reference sample and multiple dilutions with replicates to achieve accurate measurements. In contrast to real-time PCR, digital PCR [1] offers an alternative and highly precise approach that eliminates the need for both a reference sample and a standard curve. In combination with pre-validated TaqMan® Assays for quantification of Illumina® libraries, the chip-based QuantStudio® 3D Digital PCR System is perfectly suited to this application.

APPLICATION NOTE QuantStudio® 3D Digital PCR System

Precise quantification of Illumina® libraries on the QuantStudio® 3D Digital PCR System

TruSeq® REV adapterTruSeq® FWD adapter Sequence of interest

RP

ProbeFP

Nextera® REV adapterNextera® FWD adapter Sequence of interest

RP

ProbeFP

Small RNA REV adapterSmall RNA FWD adapter Sequence of interest

RP

ProbeFP

Library construction

Library quantification

Cluster generation and

sequencingData analysis

Figure 1. Workflow incorporating the QuantStudio® 3D Digital PCR System to accurately quantify Illumina® NGS libraries.

Illumina® library quantification on the QuantStudio® 3D Digital PCR System Assay designWe have designed three assays for the precise quantification of Illumina® TruSeq® DNA/RNA libraries, Nextera® libraries, and TruSeq® small RNA libraries, respectively (see “Ordering information” for a list of assay IDs and additional catalog information). TaqMan® assays for Illumina® library quantification are designed to span both the P5 (forward primer, FP) and P7

(reverse primer, RP) adapters (Figure 2), limiting quantification to only those constructs that contain both adapters. The approach described here is not unique to Illumina® NGS library quantification—TaqMan® Assays may be individually designed to quantify other NGS libraries, provided the probe and primer sequences are complementary to the appropriate adapter sequences used to generate the library [2]. Once the assay has been designed, it can be used directly in the QuantStudio® 3D Digital PCR workflow.

Figure 2. Schematic showing TaqMan® Assay designs for three Illumina® libraries. All assays are composed of a forward primer (FP), reverse primer (RP), and TaqMan® probe complementary to the Illumina® library adapter sequences.

QuantStudio® 3D Digital PCR System workfl owQuantifying NGS libraries on the QuantStudio® 3D Digital PCR System is a simple process (Figure 3). The fi rst step in the workfl ow is to dilute the library so that, on average, there are between 0.1 and 1.7 copies per reaction (Figure 4). If the dilution is outside of this range, the level of precision around the measurement will be diminished. As a general guideline, we recommend adjusting all libraries to 2 nM as determined by the Qubit® 2.0 Fluorometer prior to diluting to 0.032 pM with 1X TE. This is equivalent to a 62,500-fold dilution.

A standard PCR reaction mix is then formulated by mixing the diluted sample with QuantStudio® 3D Digital PCR Master Mix and TaqMan® Assay (Table 1). The

Figure 3. The QuantStudio® 3D Digital PCR System workfl ow for NGS library quantifi cation.

Figure 4. Copies per reaction (circled in red) for each sample, as obtained from the QuantStudio® 3D AnalysisSuite™ Cloud Software.

reaction mix is loaded onto a QuantStudio® 3D Digital PCR Chip and the chip is assembled. The sealed chip is amplifi ed on a dual fl at block thermal cycler (GeneAmp®

PCR System 9700 Dual Flat Block Thermal Cycler or ProFlex™ 2 x Flat Block Thermal Cycler) with the thermal cycling conditions shown in Table 2, and the results are read on a QuantStudio® 3D Digital PCR Instrument. Lastly, the data is visualized using QuantStudio® 3D AnalysisSuite™ Cloud Software. Based on an initial reading, dilutions may need to be adjusted above or below 62,500-fold should the resulting value not fall within the digital range for a particular library. For a detailed description of running a digital PCR experiment, please refer to the QuantStudio® 3D Digital PCR System User Guide [3].

Sealed system

Dilute Mix Load Amplify Read

Sample Target Dilution Chip Copies/rxn

TruSeq® small RNA library VIC® 1 to 7.5 x 105 B31N50_140401_175458.eds 0.352

Nextera® library VIC® 1 to 1.45 x 106 B323Y7_140401_175238.eds 0.412

TruSeq® DNA/RNA library FAM™ 1 to 1.5 x 106 B3KYC1_140401_174941.eds 1.13

ˇ ˇ ˇ ˇ ˇ

Table 1. Volumes of reagents necessary to set up a digital PCR experiment.

Component Volume (µL)

QuantStudio® 3D Digital PCR Master Mix (2X) 7.25

TaqMan® Assay (20X) 0.73

Nuclease-free water 4.52

Diluted sample 2

Total 14.5

Table 2. Thermal cycling conditions.*

Temperature Time Phase

96°C 10 min Hold

55°C 2 min39 cycles

98°C 30 sec

55°C 2 min Hold

10°C ∞ Hold* When using the ProFlex™ 2 x Flat Block Thermal Cycler, select the QuantStudio® 3D protocol template and edit the annealing temperature in accordance with the recommendation in the table above.

Interpretation of resultsThe libraries tested in this study represent three different Illumina® library types: TruSeq® DNA/RNA libraries, Nextera® DNA libraries, and TruSeq® small RNA libraries. The insert sizes in the library ranged from 150 to 500 bp as determined by a Fragment Analyzer™ system. The same library quantifi cation workfl ow, as described above, was used for all library types tested.

Data QCIntegral to the QuantStudio® 3D Digital PCR System workfl ow is an assessment of data quality, where lower-quality data is automatically fl agged on the QuantStudio®

3D Digital PCR Instrument. Any fl agged data can then be further reviewed in QuantStudio® 3D AnalysisSuite™

Cloud Software to confi rm fi nal quality. To assess the fi nal quality, a review of two criteria is recommended:

• Image view: The “Chip view” functionality found on the “Review Quality” tab is used to inspect the chip image for artifacts that may impact the fi nal result of the experiment. The chip view can be toggled between “Color by Quality” (Figure 5A) and “Color by Call” (Figure 5B). The software automatically fi lters out wells with lower-quality scores as displayed in the chip image heat map. Note that, although not recommended for most chips, the quality score threshold applied can be manually adjusted to increase

or decrease the number of wells fi ltered from the analysis. “Color by Quality” confi rms loading uniformity and image quality, while “Color by Call” confi rms a random distribution of amplifi ed wells across the chip.

• Histogram view: The histogram plot (Figure 5C) shows the discrimination between the non-amplifi ed and amplifi ed populations and confi rms that the threshold separating the populations has been correctly set by the software. If the software has not accurately set the threshold, this can be manually adjusted by the user.

The images shown in Figure 5 are for a successful experiment in which all QC metrics meet the desired outcome.

Figure 5. Views for assessing data quality in the Absolute Quantifi cation application module of QuantStudio® 3D AnalysisSuite™ Cloud Software. (A) Image from “Chip view” depicting color by quality, indicating the quality of loading on a chip. This particular chip is classifi ed as high quality due to the uniformity of the fi lled wells across the chip. (B) Image from “Chip view” depicting color by calls, which shows the distribution of amplifi ed (VIC® dye–labeled probe was used in this example) and non-amplifi ed wells. Note that a random distribution of wells across the chip is ideal. A non-random pattern could indicate possible loading issues or leaking of the immersion fl uid from the chip. (C) The “Histogram view” has two cluster populations. The yellow population corresponds to non-amplifi ed wells with lower fl uorescence compared with the amplifi ed population in red. The wide separation of the two populations is indicative of good discrimination.

A. Chip view B. Chip view

C. Histogram view

Non-amplifi ed wells

Amplifi ed wells

2.00E+9

1.50E+9

1.00E+9

5.00E+9

0

Copi

es/µ

L

Sample-target

FAM™ Nextera® FAM™ TruSe...NA/RNA FAM™ small RNA

Absolute quantificationPrior to determining the final library concentration, data must be adjusted to take into consideration the degree of sample dilution made prior to chip loading. This process is simplified in the QuantStudio® 3D AnalysisSuite™ Cloud Software by entering the dilution factor for each sample in the “Define Chips” tab. To correctly calculate the dilution factor for each sample, two components must be taken into account: (1) the sample dilution prior to preparing the reaction mix, and (2) the dilution that occurs when the reaction mix is formulated.

For example, if your sample was originally diluted 1 to 62,500 and 2 µL of the diluted sample was added to a total of 14.5 µL, then a dilution factor of 1 to 453,125 (Figure 6) should be entered into the software.

2x Master Mix20x assaySample = 2 µLNuclease-free waterTotal = 14.5 µL

Sample dilution

Dilution factor = Sample dilution x Dilution on the chip

Dilution factor to enter into software 1 to 453,125

= 62,500 x (14.5/2) = 453,125

Dilution on the chip

1 to 62,500

Figure 6. Sample calculation of a dilution factor.

Figure 7. Calculated copies/µL of various library types by QuantStudio® 3D AnalysisSuite™ Cloud Software. Error bars indicate 95% confidence level for each sample.

The QuantStudio® 3D AnalysisSuite™ Cloud Software calculates a concentration in copies/µL and graphically presents the data in the “Results” section of the “See Results” tab (Figure 7). The software provides a 95% confidence range for each sample concentration as calculated by Poisson statistics.

The final step in the quantification process is to convert the copies/µL provided by the QuantStudio® 3D Digital PCR System to a molar concentration as required for the NGS workflow. Go to lifetechnologies.com/dpcrlibquantcalc to download a calculator that converts copies/μL to nM.

Correlation between digital PCR quantification and cluster densitiesOnce the library molarity (nM) has been determined, an appropriate dilution of the library is necessary for optimal cluster generation using either on-board cluster generation or a cBot. The optimal library concentration corresponds to the library dilution that results in the appropriate cluster density on the Illumina® sequencer. Samples that fall within the recommended cluster density range (typically around 600,000–700,000 raw clusters/mm2) generally produce higher-quality data and higher outputs. Samples that are underclustered produce proportionately fewer reads, and although are generally high quality, produce lower outputs. Samples that are overclustered lead to lower pass filter rates, which leads to overall lower-quality sequencing reads and thus lower outputs.

Figure 9. Correlation of QuantStudio® 3D Digital PCR data with Illumina® cluster density data. (A) Graph depicting the concentration (nM) for fi ve Illumina® TruSeq® RNA libraries as determined by the QuantStudio® 3D Digital PCR System. The expected concentration for all fi ve libraries is 10 nM, where libraries 2 and 3 had slightly lower conentrations at 6.3 and 6.4 nM and library 4 had the highest concentration at 11.3 nM. (B) Cluster densities for the same libraries, generated by the Illumina® HiSeq® 2500 system. Libraries 2 and 3 had the lowest cluster densities and library 4 had the highest cluster density, which correlates well with the digital PCR concentrations obtained.

A

B Total clusters Pass fi lter clusters

12

10

8

6

4

2

01 2 3 4 5

Sample

Conc

entr

atio

n (n

M)

To control for variability that might be introduced during the library dilution step prior to digital analysis, a modifi ed approach to the method described above incorporates an internal positive control (IPC). This modifi cation eliminates the need to convert a copies/µL concentration into a nM concentration before proceeding to the cluster generation step of the Illumina® NGS workfl ow. Due to large upfront sample dilutions needed prior to digital PCR analysis, comparing the quantifi cation of a library to an IPC ensures that inaccurate dilutions will not adversely affect quantifi cation. For example, if the ratio between a FAM™ dye–labeled library and a VIC® dye–labeled IPC is 2.2 and the VIC® dye–labeled IPC has a known concentration of 1 nM, then the FAM™ dye–labeled library has a concentration of 2.2 nM (Figure 8). Refer to the IPC supplemental protocol for more information.

Figure 8. Example of using an internal control at a known concentration (VIC® dye–labeled IPC) to help determine the concentration of an unknown sample (FAM™ dye–labeled library).

1,200

1,000

800

600

400

200

01 2 3 4 5

Lane

Den

sity

(K/m

m2 )

96 96 96 96 96

VIC® reference ~1 nM, therefore FAM™ library ~2.2 nM

Ratio of 2.2

Copies/μL

Copies/μL

To confirm the predictive value of a digital PCR measurement, final cluster densities were evaluated on an Illumina® HiSeq® 2500 system from five Illumina® TruSeq® RNA libraries (Figure 9). No adjustment was made based on any prior knowledge of concentration. As expected, the measured concentrations established by digital PCR correlated well with the final cluster densities for all five libraries. This supports the use of the QuantStudio® 3D Digital PCR System for measurements to adjust library concentration prior to the critical cluster generation step of the Illumina® NGS workflow.

ConclusionsPrecise library quantification is a critical step prior to cluster generation. Inclusion of too much or too little library into the flow cell prior to cluster generation leads to suboptimal sequencing yields. We presented a simple digital PCR approach using carefully designed TaqMan® Assays to precisely and accurately quantify TruSeq® DNA/RNA libraries, Nextera® libraries, and TruSeq® small RNA libraries. The QuantStudio® 3D Digital PCR System is ideally suited for the quantification of Illumina® NGS libraries for the following reasons:

• Direct measurement of molecules that contain both library adapters is obtained, as compared to current detection methods

• Absolute concentration measurements are provided that help maximize success in obtaining optimal cluster densities and overall sequencing yield

• Highly affordable system and consumables, with possible cost savings when considering the consequences of resequencing samples due to suboptimal clustering

• Precise quantification can be obtained without the need for a reference sample

References1. Pohl G, Shih IM (2004) Principle and applications of digital PCR. Expert

Rev Mol Diagn 4(1):41–47.2. Precise quantification of Ion Torrent™ libraries on the QuantStudio®

3D Digital PCR System. (2014) Application note available at lifetechnologies.com (Pub. No. CO28554).

3. QuantStudio® 3D Digital PCR System User Guide. Pub. No. MAN0007720, Rev. B.

AcknowledgmentWe would like to acknowledge the work of Dr. Peter Schweitzer, Genomics Facility Director, Cornell University, for his contribution to this work and application note.

Find out more at lifetechnologies.com/quantstudio3dFor Research Use Only. Not for use in diagnostic procedures. © 2014 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a registered trademark of Roche Molecular Systems, Inc., used under permission and license. Illumina, HiSeq, MiSeq, TruSeq and Nextera are registered trademarks of Illumina, Inc. Bioanalyzer is a registered trademark of Aglient Technologies, Inc. Fragment Analyzer is a trademark of Advanced Analytical Technologies, Inc. CO35377 1014

Ordering information

Product Cat. No.

QuantStudio® 3D Digital PCR System Package—includes:* A25581**

QuantStudio® 3D Digital PCR Instrument 1 instrument 4489084

QuantStudio® 3D Digital PCR Chip Loader 1 loader 4482592

ProFlex™ 2 x Flat PCR System 1 thermal cycler 4484078

QuantStudio® 3D Digital PCR Chip Adapter Kit 1 kit 4485513

QuantStudio® 3D Digital PCR UV Sealing Kit 1 kit 4488475

QuantStudio® 3D Digital PCR 20K Chip Pack(includes consumables)

12 chips per pack(package includes 8 packs)

4485507

QuantStudio® 3D Digital PCR Master Mix1.5 mL(package includes 1 tube)

4482710

QuantStudio® 3D Digital PCR Tilt Base for ProFlex™ Thermal Cycler A24898

Additional items

QuantStudio® 3D Digital PCR Master Mix 5 mL 4485718

QuantStudio® 3D AnalysisSuite™ Server 1 server system 4489085

Assays

TaqMan® Gene Expression Assay for TruSeq® DNA/RNA Library Quantification, 20X, Assay ID: Ac04364396_a1 4331182

TaqMan® Gene Expression Assay for Nextera® Library Quantification, 20X, Assay ID: Ac04347696_a1 4331182

TaqMan® Gene Expression Assay for Small RNA Library Quantification, 20X, Assay ID: Ac04347697_a1 4331182

*Part numbers listed in bundle are for individual components. ** Cat. No. A25581 is for all regions except Europe, the Middle East, and Africa (EMEA). Please use Cat. No. A25606 for customers residing in EMEA.

Package components are slightly different. Please check with your regional sales representative for details.