Droplet Digital PCR Droplet Digital™ PCR Applications Guide
Table of Contents iii Chapter 1 Droplet Digital™ PCR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 QX100/QX200 Workflow. . . . . . . . . . . . . .
Table of Contents Statistics of ddPCR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Copies per Microliter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Copies per Droplet. . . . . . . . . . . . . . . . . . . . . . . . . . . .
Table of Contents One-Step RT-ddPCR Kit for Probes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Data Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ddPCR Gene Expression Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 Droplet Digital™ PCR Introduction Droplet Digital polymerase chain reaction (ddPCR™) was developed to provide high-precision, absolute quantification of nucleic acid target sequences with wide-ranging applications for both research and clinical diagnostic applications. ddPCR measures absolute quantities by counting nucleic acid molecules encapsulated in discrete, volumetrically defined water-in-oil droplet partitions.
Droplet Digital™ PCR ddPCR has the following benefits for nucleic acid quantification: ■■ ■■ ■■ ■■ Unparalleled precision — the massive sample partitioning afforded by ddPCR enables small fold differences in target DNA sequence between samples to be reliably measured Increased signal-to-noise — enrich for rare targets by reducing competition that comes from high-copy templates Removal of PCR efficiency bias — error rates are reduced by removing the amplification efficiency reliance of PCR, enabling acc
Droplet Digital™ PCR Droplet Generation Before droplet generation, ddPCR reactions are prepared in a similar manner as real-time PCR reactions that use TaqMan hydrolysis probes labeled with FAM and HEX (or VIC) reporter fluorophores, or an intercalating dye such as EvaGreen. ddPCR must be performed with the proprietary reagents developed specifically for droplet generation by Bio-Rad.
Droplet Digital™ PCR PCR Amplification Droplets are transferred to a 96-well plate for PCR in a thermal cycler. We recommend the C1000 Touch™ thermal cycler with 96–deep well reaction module for PCR (Figure 1.5). This high-performance thermal cycler has excellent temperature uniformity and settling across all 96 wells to help ensure successful PCR. Fig. 1.5. The C1000 Touch thermal cycler provides robust performance for ddPCR experiments.
Droplet Digital™ PCR Droplets are spaced out individually for fluorescence reading by the droplet reader (Figure 1.7). Fluorescence in two channels is then measured for individual droplets. Fig. 1.7. Separating individual droplets in the QX100 droplet reader. Positive droplets, which contain at least one copy of the target DNA molecule, exhibit increased fluorescence compared to negative droplets (Figure 1.8). Fig. 1.8. Fluorescence readings are measured for each droplet in two channels.
Droplet Digital™ PCR Droplet Digital PCR data from a duplex experiment in which two targets are PCR amplified can also be viewed in a 2-D plot in which channel 1 fluorescence (FAM) is plotted against channel 2 fluorescence (HEX or VIC) for each droplet (Figure 1.10). 10,000 9,000 8,000 Channel 1 amplitude 7,000 6,000 5,000 4,000 3,000 2,000 1,000 0 0 1,000 2,000 3,000 4,000 5,000 6,000 7,000 Channel 2 amplitude Fig. 1.10. 2-D plot of droplet fluorescence.
Droplet Digital™ PCR Emerging Applications of Droplet Digital PCR Sample partitioning allows the sensitive, specific detection of single template molecules as well as precise quantification. It also mitigates the effects of target competition, making PCR amplification less sensitive to inhibition and greatly improving the discriminatory capacity of assays that differ by only a single nucleotide. Digital PCR offers the benefits of absolute quantification and greatly enhanced sensitivity.
Droplet Digital™ PCR p= Sample 1 Sample 2 Sample 3 Sample 4 No targets Low concentration Medium concentration High concentration 0 positive 143 total p= 6 p= 143 Poisson corrected 6.2/143 34 Poisson corrected 38/143 70 143 Poisson corrected 96/143 4.5 90,000 4 80,000 3.5 70,000 3 60,000 2.5 50,000 2 40,000 1.5 30,000 1 20,000 0.5 10,000 Copies of target/20 µl sample 100,000 5 Target copies/droplet p= 143 0 0 0 0.2 0.4 0.6 0.
Droplet Digital™ PCR A A02 Channel 2 amplitude 8,000 B02 C02 D02 E02 F02 G02 H02 A02 8,000 B02 C02 D02 E02 F02 G02 H02 7,000 7,000 7,000 6,000 6,000 6,000 5,000 5,000 5,000 4,000 4,000 3,000 3,000 Low threshold C02 D02 E02 F02 G02 H02 3,000 High threshold 2,000 2,000 1,000 1,000 1,000 0 0 20,000 B02 4,000 Medium threshold 2,000 0 A02 8,000 40,000 60,000 80,000 100,000 120,000 Event number 0 0 20,000 40,000 60,000 80,000 100,000 120,000 Event number
Droplet Digital™ PCR Concentration, copies/µl A 100,000 10,000 1,000 1,030 1,020 1,050 1,060 100 10 999 481 1,040 247 952 118 61 31.5 17.3 7.58 1,060 6,360 3,950 2,040 983 990 986 1,080 1,000 1 0.1 0.
2 Designing Droplet Digital™ PCR Experiments Assay Design for Droplet Digital PCR As with any PCR-based technology, assay design and sample preparation are important for obtaining high-quality data. Before running a Droplet Digital (ddPCR™) experiment, know the goal or possible expected outcomes of the experiment because different types of experiments require different controls, sample preparation, amounts of DNA or RNA, and data analysis.
Designing Droplet Digital™ PCR Experiments When designing primers for a target sequence, follow these guidelines: ■■ ■■ ■■ Design primers that have a GC content of 50–60% Strive for a Tm between 50 and 65°C. One way to calculate Tm values is by using the nearest-neighbor method. Use the Tm calculator at http://www.basic.northwestern.edu/ biotools/oligocalc.
Designing Droplet Digital™ PCR Experiments R Q During annealing, the hydrolysis probe binds to the target sequence Extension R Q During extension, the probe is partially displaced and the reporter is cleaved. The free reporter fluoresces R Q R Reporter Q Quencher Fig. 2.1. In TaqMan assays, hydrolysis probes are hydrolyzed by Taq polymerase. When designing probes, use the following guidelines: ■■ ■■ ■■ ■■ ■■ ■■ ■■ The probe sequence must be chosen between the two primers of the amplicon.
Designing Droplet Digital™ PCR Experiments Fig. 2.2. Main tab in Primer3Plus. In the Main window, paste your target DNA sequence in the “Paste source sequence below” field. We recommend the following changes to the default settings when designing ddPCR assays: ■■ ■■ ■■ In the General Settings window, change “Concentration of divalent cations” to 3.8, “Concentration of dNTPs” to 0.
Designing Droplet Digital™ PCR Experiments Fig. 2.3. Assay design output. Several important design features are not addressed by Primer3Plus (or the Primer3 MIT site). ■■ ■■ ■■ To ensure primer specificity, use tools such as BLAST (Basic Local Alignment Search Tool), hosted at the National Center for Biotechnology Information (NCBI), either in the “traditional” general search form (www.ncbi.nlm.nih.gov/BLAST) or a form tailored specifically to check that PCR primers (http://www.ncbi.nlm.nih.
Designing Droplet Digital™ PCR Experiments For best results, restriction digestion of your DNA sample outside of the amplicon region is recommended. We have extensively investigated the use of endonucleases for fragmentation and found that a wide range of enzymes with 4-base and 6-base recognition sites perform satisfactorily for this purpose. The benefits of predigestion can be achieved with a wide range of enzyme concentrations.
Designing Droplet Digital™ PCR Experiments The example is relevant to any gene that is present at the normal rate of 2 copies/diploid genome, such as RPP30, and provides a basis for a digital screening experiment to determine the optimal digital range.
Designing Droplet Digital™ PCR Experiments Fig. 2.4. Loaded DG8 cartridge. Note: Each DG8 cartridge generates eight wells of droplets. Any unused wells on the cartridge must be filled with 1x ddPCR buffer control. The Bio-Rad ddPCR supermixes have been formulated specifically to work with the droplet chemistry. Altering the components used in the QX100 droplet generator or using a different supermix will negatively impact results.
Designing Droplet Digital™ PCR Experiments After heat sealing, place the PCR plate in a thermal cycler for PCR using the following guidelines. ■■ ■■ ■■ ■■ Use a recommended thermal cycling protocol Use a 2.5°C/sec ramp rate to ensure each droplet reaches the correct temperature for each step during the cycling 40 cycles of PCR is enough for an optimized ddPCR assay. Do not exceed 50 cycles After PCR, the plate can be left in the thermal cycler overnight at 10°C or stored at 4°C.
Designing Droplet Digital™ PCR Experiments Droplet Reading Following PCR amplification of the nucleic acid target in the droplets, place the PCR plate in a QX100 or QX200 droplet reader. The droplet reader and QuantaSoft software count the PCR-positive and PCR-negative droplets to provide absolute quantification of target DNA.
Designing Droplet Digital™ PCR Experiments Fig. 2.7. Setting thresholds in a 1-D plot. Note: Use 1-D plots for temperature gradient experiments and wells containing single assays. For all experiments involving duplex assays, set thresholds in the 2-D plot view (Figure 2.8). For correct quantification of a single-color experiment, use the thresholding tools to ensure correct designation of the populations as negatives (gray) and positives (blue or green).
Designing Droplet Digital™ PCR Experiments Fig. 2.8. Setting thresholds in a 2-D plot. For correct quantification of a duplex experiment, use the thresholding tools to ensure correct designation of the droplet populations as double negative (gray), FAM positive (blue), VIC/HEX positive (green), and double positive (brown = positive for FAM and HEX/VIC in the same droplet). There is an autoanalysis tool as well as several different manual thresholding tools including a “free-draw” tool.
Designing Droplet Digital™ PCR Experiments PCR Optimization Using Thermal Gradients Optimizing the annealing temperature of your PCR assay is one of the most critical parameters for reaction specificity. Setting the annealing temperature too low may lead to amplification of nonspecific PCR products. On the other hand, setting the annealing temperature too high may reduce the yield of a desired PCR product.
Designing Droplet Digital™ PCR Experiments ddPCR Using the QX200 System and EvaGreen dsDNA Dye The QX200 system can measure amplified DNA using Bio-Rad’s QX200 ddPCR EvaGreen supermix, template, and a pair of primers. EvaGreen dsDNA binding dye is similar to SYBR® Green in that it fluoresces upon binding double-stranded DNA (Figure 2.11). DNA Inactive form of EvaGreen Active form of EvaGreen EvaGreen-DNA complex Fig. 2.11. EvaGreen dye binds to dsDNA via a “release-on-demand” mechanism.
Designing Droplet Digital™ PCR Experiments In Figure 2.13, a gradient of annealing/extension temperatures is shown for three amplicons with different lengths: A, B, and C correlate to 200, 99, and 62 bp amplicons, respectively. The experiment demonstrates the effect amplicon length has on fluorescence amplitude as well as how annealing/extension temperature affects amplitude and the ability to resolve products with different lengths based on amplicon size.
Designing Droplet Digital™ PCR Experiments EvaGreen and Gene Expression Measuring gene expression using ddPCR and EvaGreen allows you to detect and quantify splice variants as well as contaminating genomic DNA. Figure 2.14 shows an experiment demonstrating that splice variants can be seen as a cluster of droplets with higher fluorescence amplitude, referred to as a super cluster.
Designing Droplet Digital™ PCR Experiments Multiplexing with EvaGreen Differences in droplet amplitude due to differences in amplicon length or optimal annealing temperature allow for multiplexing in a single well. Figure 2.15 shows a 2-D fluorescence plot containing two amplicons, where differences in amplitude are observed due to differences in optimal annealing temperature. Primer set 1 has an optimal Tm of 63˚C, therefore droplets having this target have relatively high fluorescence amplitudes at 63˚C.
3 Absolute Quantification and the Statistics of Droplet Digital™ PCR Running Absolute Quantification Experiments Absolute quantification (ABS) is fundamental to all Droplet Digital PCR (ddPCR™) applications. Partitioning template DNA into uniform droplets enables highly quantified measurements of target DNA using the QX200™ or QX100™ Droplet Digital PCR system and the appropriate automatic statistical analysis in QuantaSoft™ software.
Absolute Quantification and the Statistics of Droplet Digital™ PCR Note: Do not exceed 5,000 copies of target/µl of the final ddPCR reaction mix.
Absolute Quantification and the Statistics of Droplet Digital™ PCR 10,000 Concentration, copies/µl 1,000 1,390 1,400 260 259 100 1,400 1,400 64.2 64.8 10 1,360 1,370 15.5 15 1,380 1,390 4.63 4.68 1 1,370 1,360 1.46 1.53 0.10 0.01 0 256 256 64 64 16 16 4 4 1 1 Sample Fig. 3.2. Sample concentrations are plotted as copies/µl from the sample. Statistics of ddPCR In ddPCR, DNA molecules and PCR reagents are partitioned into droplets.
Absolute Quantification and the Statistics of Droplet Digital™ PCR Copies per Microliter QuantaSoft software provides concentration results in copies of target per microliter (copies/µl). The dynamic range of ddPCR extends from fewer than 0.25 copies/µl to more than 5,000 copies/µl. In many cases, the fundamental quantity of interest to a user is the number of copies of target in the starting sample. The following example shows how copies/µl is converted to copies in the starting material.
Absolute Quantification and the Statistics of Droplet Digital™ PCR Low Concentration Example When there are far fewer molecules than partitions (for example, 500 molecules or less in 20,000 partitions), it is relatively easy to see how the ddPCR approach enables accurate quantification. Figure 3.3 shows a sample that contains six target DNA molecules.
Absolute Quantification and the Statistics of Droplet Digital™ PCR Intermediate Concentration Example Consider the case in which there are 5,000 target molecules in 20,000 droplets (5,000 targets in 20 µl = 250 copies/µl = 0.25 CPD). Random partitioning of target molecules into droplets will lead to some droplets with 2, 3, or even 4 copies, and correspondingly more than 75% of the droplets will have zero copies. Poisson statistics tells us exactly how many droplets to expect in each category. Table 3.
Absolute Quantification and the Statistics of Droplet Digital™ PCR Even at an average of 5 copies/droplet (the upper end of the recommended loading range), we expect to see about 134 empty droplets in a total of 20,000 droplets (data not shown). Looking across the Whole Concentration Range Figure 3.4 shows the number of droplets with 0, 1, 2, 3 (and so on) copies of the target DNA at different DNA concentrations.
Absolute Quantification and the Statistics of Droplet Digital™ PCR Formula for Calculating Concentration The formula used by QuantaSoft software to calculate concentration is: Concentration = –In ( Nneg ) /Vdroplet N Derivation of Concentration Formula The Poisson distribution gives probability Pr(n) that a droplet will contain n copies of target if the mean number of target copies per droplet is C: Cne–c n! Pr(n) = Inputting n = 0 gives the probability that a droplet will be empty for a given value of
Absolute Quantification and the Statistics of Droplet Digital™ PCR Errors in ddPCR Two types of errors are reported by QuantaSoft software: technical errors (Poisson errors) and total errors. Technical errors (Poisson errors): a measurement error based on known properties of the system that can be calculated based on a single well or by pooling all the droplets from multiple wells. One of the assumptions in this error calculation is that the sample in a ddPCR well is a subsample from a larger whole.
4 Copy Number Variation Analysis Overview Analysis of copy number (CN) involves determining the number of copies of a given target locus with respect to an invariant reference locus. An alteration in copy number state with respect to the reference locus is copy number variation (CNV). CNV could be a deletion or duplication of a locus with respect to the number of copies of the reference locus (and hence genomes) present in the cell.
Copy Number Variation Analysis 100 Copy number resolution: N vs. N–1, % 90 80 70 60 50 40 30 20 10 0 0 2 4 6 8 10 12 14 16 18 Copy number, N 20 22 24 26 28 30 Fig. 4.1. Discrimination between consecutive CN states is more difficult at higher order copy numbers. The massive partitioning of a CNV Droplet Digital™ PCR (ddPCR™) reaction across up to 20,000 droplets enables the fine quantitative discrimination required to resolve consecutive copy number states beyond CN 3.
Copy Number Variation Analysis CNV Calculations CNV analysis by ddPCR involves quantification of target and reference loci through the use of duplex target and reference assays. In QuantaSoft™ software, copy number is determined by calculating the ratio of the target molecule concentration to the reference molecule concentration, times the number of copies of reference species in the genome (usually 2).
Copy Number Variation Analysis Table 4.1. Detection of CN variant cells in a heterogeneous sample. Sensitivity Required for CN Determination in a Heterogeneous Sample Diploid Copy Number Cells with CN Alteration, % Wild Type 2 Amplified 3 10 2 10 40 2 50 240 1 0.1 0.01 Discrimination Needed, % 5 2 3 0.5 2 10 4 2 50 24 2 3 0.05 2 10 0.4 2 50 2 2 3 0.005 2 10 0.040 2 50 0.
Copy Number Variation Analysis Running a CNV Assay Select Copy Number Variation (CNV) as the experiment type in QuantaSoft software when loading wells. Double click on the experiment name in the main software window to set the ploidy for the reference (Figure 4.3). Fig. 4.3. Setting the ploidy for the CNV reference. Note: R2 means 2 copies/genome (diploid) or 1 copy/haploid genome. If CNV experimental type is not selected for your wells, the CNV tab will not be available.
Copy Number Variation Analysis Considerations in planning a restriction digestion. ■■ Do not cut the target or reference amplicon ■■ Choose a methylation-insensitive enzyme ■■ ■■ ■■ ■■ Read the chosen restriction enzyme FAQs on the manufacturer’s website. They often describe known issues such as star activity For most assays, a fragment size of 5 kb or less works fine. This can typically be achieved with a 4-cutter or 6-cutter enzyme.
Copy Number Variation Analysis Lists of recommended restriction enzymes for CNV ddPCR are provided in Tables 4.2 and 4.3. Conditions for a typical restriction enzyme digestion are in Table 4.4. For more information, visit the New England Biolabs, Inc. website (www.neb.com). Table 4.2. Recommended restriction enzymes for CNV ddPCR (most-preferred 4-cutters). Restriction Enzyme CviQI Sequence G/TAC Digestion Buffer (old) NEBuffer 2, 3, BSA Digestion Buffer (new) 3.
Copy Number Variation Analysis DNA Loading for Lower-Order CN Analysis (diploid CN <10) For most routine CNV ddPCR applications, where a diploid target CN is expected to be 10 or less, approximately 0.2–1.0 reference gene copies per droplet (CPD) of DNA sample should be loaded per well. This corresponds to 10–66 ng of human genomic DNA per well.
5 Rare Mutation and Sequence Detection Overview Droplet Digital™ PCR (ddPCR™) enables detection and analysis of nucleic acids at a level of sensitivity and precision beyond the capabilities of previous methods. Applications that focus on the lower limits of nucleic acid detection can be separated into two classes: ■■ Rare mutation detection ■■ Rare sequence detection Rare mutation detection (RMD) and rare sequence detection (RSD) can be classified according to their assay components.
Rare Mutation and Sequence Detection Measurement Units Examples Assay Components Assay Schematic Rare Mutation Detection Ratio (% or a/[a+b]) ■■ umor biopsy: 0.
Rare Mutation and Sequence Detection Rare Mutation Detection Rare mutation detection occurs when a biomarker exists within a background of a highly abundant counterpart that differs by only a single nucleotide. Many methods for mutation analysis have poor selectivity and fail to detect mutant sequences with abundances of less than one in 100 wild-type sequences (Scott 2011, Benlloch et al. 2006, Whitehall et al. 2009).
Rare Mutation and Sequence Detection RMD Experiment Considerations The first consideration for low-level detection is the amount of DNA available. If 1 mutant in 100,000 wild-type sequences, or 0.001%, is to be detected, then statistically at least 300,000 haploid genomes must be screened. For human DNA this is 1 µg of DNA. The challenge for RMD assay development is that it must discriminate between two highly similar sequences, one of which is significantly more abundant than the other.
Rare Mutation and Sequence Detection A A03 14,000 B03 C03 D03 E03 F03 G03 H03 A04 B04 C04 D04 E04 F04 G04 H04 Channel 1 amplitude 12,000 10,000 8,000 6,000 4,000 2,000 0 0 50,000 100,000 Event number 150,000 100% wild-type DNA B A06 14,000 B06 C06 D06 E06 F06 50% mutant/wild-type DNA G06 H06 A07 B07 C07 D07 E07 F07 G07 H07 Channel 1 amplitude 12,000 10,000 8,000 6,000 4,000 2,000 0 0 50,000 100% wild-type DNA 100,000 Event number 150,000 200,000 50% mutant/wild-
Rare Mutation and Sequence Detection Figure 5.5 shows a 2-D plot of a single well with an assay for SNP KRAS G12V that indicates no false positives at 5,000 copies/µl. This assay can detect 0.001% mutant, or 1 mutant in 100,000 wild-type targets. 8,000 Channel 1 amplitude 7,000 6,000 5,000 4,000 3,000 2,000 1,000 0 0 1,000 2,000 3,000 4,000 5,000 Channel 2 amplitude 6,000 7,000 8,000 9,000 Fig. 5.5.
Rare Mutation and Sequence Detection In Figure 5.
Rare Mutation and Sequence Detection Additional wells may be screened to ensure detection of more than one positive droplet. For ddPCR, the ability to merge multiple wells into a meta-well provides more experimental flexibility to tune the experiment to the required LoD. The number of cells or amount of DNA needed to screen a given number of background molecules is described in Table 5.1. The number of wells needed is a conservative estimate. The row in bold reflects the best performance in one well.
Rare Mutation and Sequence Detection We recommend always running negative controls to monitor for all possible sources of false-positive droplets, given the risk of laboratory contamination when working with templates and amplicons. With an extremely sensitive technology such as ddPCR, we recommend running enough negative sample controls as part of every experiment so that you can demonstrate that the probability of calling a true negative sample incorrectly is below a certain threshold.
Rare Mutation and Sequence Detection RSD Experimental Strategies Some RSD applications require reliable quantification of rare sequences while others require detection of a rare sequence. This difference dictates the lower bounds of sensitivity for a given assay and application. For RSD, the LoD can be defined either in terms of the total volume of material analyzed or in terms of the number of copies of some type of background DNA. In RSD, absolute quantification of the target sequence is often required.
Rare Mutation and Sequence Detection Case 2: Quantification with Respect to Second DNA Sequence To detect a very low target concentration in a high background sample, for example 1 copy of virus/100,000 peripheral blood mononuclear cells (PBMCs), the sample can be analyzed at two different concentrations (Figure 5.9). The high-concentration wells provide sensitive detection of the rare sequence while the low-concentration wells enable quantification of the background sequence.
Rare Mutation and Sequence Detection In the example given, the measurement error (CV) is introduced at the time of subsampling (at the time of the blood draw), not by the measurement system. Poisson statistics dictate that if N target molecules are measured in a sample, the standard deviation of the measurement is roughly the square root of N. In this example, if 100 copies of virus are present in the sample, the standard deviation of the measurement is 10 and the CV is 10/100 = 10%.
6 Gene Expression Overview Reverse transcription quantitative PCR (RT-qPCR) is a commonly used method in gene expression studies. It is straightforward, sensitive, and has a wide dynamic range. There are two types of approaches for the RT-qPCR reaction: one-step and two-step RT-qPCR. One-Step RT-qPCR One-step RT-qPCR simplifies the reaction setup by combining the first-strand cDNA synthesis (reverse transcription) and qPCR in one mixture.
Gene Expression Similar to RT-qPCR, reverse transcription Droplet Digital™ PCR (RT-ddPCR) approaches can also use a one-step or two-step protocol. Choose your approach based on your experimental purpose. Two-Step Reverse Transcription ddPCR Obtain RNA Use a commercial kit to extract RNA. Store at 100 ng/µl in 1/10 TE buffer (0.1x TE) or other appropriate buffer at –80°C. Generate cDNA Generate cDNA according to standard protocols.
Gene Expression The one-step RT-ddPCR kit for probes is formulated for efficient and sensitive reverse transcription over a wide linear dynamic range of input RNA for ddPCR. Use a commercial kit to extract RNA, then store RNA at 100 ng/µl in 1/10 TE buffer (0.1x TE) or other appropriate buffer at –80˚C. A unique hot-start reverse transcriptase enables convenient reaction setup.
Gene Expression ddPCR Gene Expression Data As shown in Figure 6.2, cDNA copies were made on three in vitro transcribed RNAs with the one-step RT-ddPCR kit for probes or two-step RT-qPCR with iScript advanced cDNA synthesis kit for RT-qPCR. The data are generally comparable on most of the assay locations. A few sites are much lower in one-step RT-ddPCR because those sites may be involved in secondary structures and the priming sites might not be accessible to the reverse transcription enzyme.
Gene Expression Figure 6.3 illustrates the determination of ERBB2 transcript levels relative to the EEF2 reference gene. The “best coverage” ERBB2 assay was tested with four fresh-frozen breast tumor RNA samples (OriGene Technologies, Inc). FAM (ERBB2) concentration, VIC (EEF2) concentration, and normalized ERBB2 to EEF2 concentrations are shown in Figure 6.3 (blue, green, and maroon, respectively). 4,000 5 3,400 4 3,130 3.04 2,400 3 1,650 1,600 1.57 1,050 1.28 800 2 1,030 1 658 514 215 0.
Gene Expression 3,410 1,000 656 Concentration, copies/µl 618 1,170 1.58 1.27 217 3,140 3.03 1,040 1,730 1,090 1.0 100 15.7 10 0.10 1.0 0.0636 0.10 0.0133 0.01 CR560536 cD CR560258 cD CR562124 cD CR561507 cD Normalized ERBB2 to GAPDH concentration Figure 6.5 is an example of normal breast tissue compared with HER2+ tissues (fresh frozen). 0.01 Ambion normal Sample Fig. 6.5. Comparison of HER2 expression levels of fresh frozen tumor versus normal tissue. Figure 6.
Gene Expression Table 6.1 shows a comparison of IHC, FISH, genomic copy number, and transcript level in clinical samples. The table shows the capability of ddPCR to determine gene copies and ERBB2 cDNA transcript levels when normalized to a reference transcript (EEF2 or GAPDH). ddPCR results are in excellent agreement with pathology results, and have the added benefit of being quantitative. Table 6.1. Comparison of clinical pathology results with ddPCR gene expression data.
7 Next-Generation Sequencing Library Analysis Overview Next-generation sequencing (NGS) systems are extremely sensitive to the quantity of library loaded in the sequencing run. Overloading frequently produces unusable data and underloading wastes reagents and time. Droplet Digital™ PCR (ddPCR™) complements NGS by offering accurate library concentration measurements and unique quality analyses that are not available with other methods. Standard methods for quantifying NGS libraries have disadvantages.
Next-Generation Sequencing Library Analysis ddPCR Quantification on Illumina TruSeq v2 Chemistry For the Illumina MiSeq and HiSeq platforms, the total possible reads is directly related to the concentration of prepared library loaded. These platforms have a narrow loading concentration range requirement for successful runs. To maximize the sequencing information from a given sequencing run, accurate measurements of library concentration must be made.
Ligate index adapter P5 Rd1 SP DNA insert Next-Generation Sequencing Library Analysis Index Rd2 SP P7 Denature and amplify for final product H FP Probe 1 Q F Probe 2 Q RP Fig. 7.2. Design of the ddPCR library quantification kit for Illumina TruSeq assay. An example of the 2-D plot of ddPCR FAM versus HEX data observed for this assay is shown in Figure 7.3.
Next-Generation Sequencing Library Analysis Droplets that appear above and below the insert population (large, diagonal cluster of increasing fluorescence) represent rare species with three or more adapters ligated to the insert (Figure 7.5). These populations can be selected in QuantaSoft™ software using the lasso function. By selecting the desirable bulk population that lies along the diagonal (green circle, Figure 7.
Next-Generation Sequencing Library Analysis 10 Total reads, millions 8 6 4 Total reads 2 PF reads Q30 reads 0 0 2 4 6 8 10 Concentration, pM 12 14 16 Fig. 7.6. Plot of the reads vs. input library concentration. Impact of input library concentration on total usable reads. Cluster density at 5 pM was approximately 800,000/mm2. PF, passing filter.
Next-Generation Sequencing Library Analysis A 800 PF reads (x 1,000) 700 600 500 400 300 200 100 L ib ra r y7 L ib ra r y8 L ib ra r y9 L ib rar y1 0 L ib ra r y1 1 L ib rar y1 2 y5 y6 ra r ra r L ib L ib y3 y4 ra r L ib y2 ra r ra r L ib L ib ra r L ib Av e rag e y1 0 Channel 2 amplitude B Library PF reads identified, % 10 Input total brain RNA 1 2 3 4 4,000 5 6 1,000 7 8 9 10 100 11 12 10 8.
Next-Generation Sequencing Library Analysis 3. Add 20 µl of TE buffer for each well used; add additional TE buffer by multiplying by the number of combined replicate wells if applicable. 4. In a fume hood, add 70 µl of chloroform for each well and cap the tube. Add additional chloroform by multiplying by the number of combined replicate wells if applicable. 5. Vortex at maximum speed for 1 min. 6. In a centrifuge, spin down at 15,500 x g for 10 min. 7.
Next-Generation Sequencing Library Analysis bp bp Ladder AF NTC SM NTC AF 1.2 SM 1 AF NTC SM NTC AF 1.2 SM 1 AF NTC SM NTC AF 1.2 SM 1 1,500 — — 1,500 850 — 700 — — 850 — 700 500 — — 500 400 — — 400 300 — — 300 200 — — 200 150 — — 150 100 — — 100 50 — — 50 15 — — 15 L 1 2 3 4 5 6 7 8 9 10 11 12 Fig. 7.9. Postrecovery analysis of ddPCR products. bp, base pair; NTC, no template control. In Figure 7.
8 Additional Applications Linkage Analysis Physical linkage of two alleles in Droplet Digital™ PCR (ddPCR™) can be thought of in at least two ways: ■■ Tandem repeats of the same sequence ■■ Proximity of two sequences (targets) physically linked on the same piece of DNA We recommend using restriction enzymes to digest the DNA in a copy number variation (CNV) study and physically separate the two target copies (in order to ensure random target distribution into droplets).
Additional Applications 5 4 Sample 1 Cut Uncut Copy number 3 Sample 2 Cut Uncut Sample 3 Cut Uncut Sample 4 Cut Uncut Sample 5 Cut Uncut 2 1 BC_19108 BC_19108 BC_19108 BC_19108-Rsal BC_19108-Rsal BC_18853 BC_19108-Rsal BC_18853 BC_18853 BC_18853-Rsal BC_18853-Rsal BC_152 BC_18853-Rsal BC_152 BC_152 BC_152-Rsal BC_152-Rsal BC_106 BC_152-Rsal BC_106 BC_106 BC_106-Rsal BC_106-Rsal BC_090 BC_106-Rsal BC_090 BC_090 BC_090-Rsal BC_090-Rsal BC_090-Rsal 0 Sample Fig. 8.1.
Additional Applications Demonstration of a milepost assay is presented in Figure 8.3, which shows the 2-D plots of FAM amplitude and VIC amplitude for an RNaseP anchor assay (VIC), and progressively farther assays on chromosome 10 for a human DNA sample. The upper left panel demonstrates a control to account for the inherent probability of two copies completely separated from each other (RNaseP, which is on chromosome 10, and an assay located on chromosome 6) randomly co-localizing in the same droplet.
Additional Applications ddPCR amplification of the cDNA generated in the RT reaction was done by adding 1.33 µl of each cDNA to a 20 µl ddPCR reaction mixture containing 1 µl of 20x TaqMan miRNA reagent (Life Technologies) specific to the miRNA of interest, 10 µl of Bio-Rad’s 2x ddPCR supermix for probes, and 7.67 µl of molecular biology–grade water. The droplets were generated, thermal cycled, and detected using standard procedures.
Additional Applications A good starting point would be to halve, or conversely, double the concentration of an assay relative to the standard final 1x concentration in order to spatially resolve the two assays in a 2-D plot of FAM and HEX amplitudes. Figure 8.5 is an assay targeting chromosome 10 of the human genome.
Additional Applications In this experiment, a single FAM assay was used (RPP30) at standard 1x final concentration and two different HEX assays at different concentrations (Chr13q3 and Chr10q1). Assay Chr10q1 was used at 1/2x standard concentration and assay Chr13q3 was used at 1x final concentration in the reaction. All three assays were present along with template in the supermix before making droplets.
9 Droplet Digital PCR Tips, Assay Considerations, and Troubleshooting ™ Assay-Dependent Cluster Shifts As with any PCR-based technology, assay design and sample preparation are important for obtaining good quality data. Before running a Droplet Digital PCR (ddPCR™) experiment, know the goal or possible expected outcomes of the experiment because different types of experiments require different controls, sample preparation, amounts of DNA or RNA, and data analysis.
Droplet Digital™ PCR Tips, Assay Considerations, and Troubleshooting such confusion, always classify the droplets of cross-reacting assays while viewing the 2-D amplitude plots. For users operating QuantaSoft™ software version 1.2.10 or earlier, appropriate droplet classification using a linear threshold may not be possible without misclassifying some droplets. Upgrading to QuantaSoft software version 1.3.2 or higher allows for proper classification using the clustering tools.
Droplet Digital™ PCR Tips, Assay Considerations, and Troubleshooting 14,000 Channel 1 amplitude 12,000 10,000 8,000 6,000 4,000 2,000 0 1,000 0 2,000 3,000 4,000 5,000 6,000 7,000 Channel 2 amplitude Fig. 9.2. Extra droplet clusters. To prevent the off-target amplification, try increasing the annealing temperature of the PCR reaction to improve specificity (Figure 9.
Droplet Digital™ PCR Tips, Assay Considerations, and Troubleshooting Positive Droplets in No Template Control Wells Digital PCR can detect very low levels of target DNA so it is important to prevent template/ amplicon contamination and to run no template controls (NTCs). Positive droplets in NTC wells that are at intensities equal to those of positive droplets in sample wells are typically caused by template or PCR product (amplicon) contamination in the reagents.
Droplet Digital™ PCR Tips, Assay Considerations, and Troubleshooting Note: The UNG approach can address contamination caused by PCR products created using ddPCR supermix for probes or one-step RT-ddPCR kit for probes; however, it will not address contamination caused by sample-source templates or PCR products created using droplet PCR supermix.
Droplet Digital™ PCR Tips, Assay Considerations, and Troubleshooting No or Few Positive Droplets If a new, never-before-tested assay fails to give positive droplets, consider the following: 1. The selected restriction enzyme may have cut within the target locus. – Recommendation: test the assay against DNA digested with a different restriction enzyme as well as undigested DNA 2. The target locus resides in a region that contains secondary structure.
Droplet Digital™ PCR Tips, Assay Considerations, and Troubleshooting ■■ ■■ ■■ ■■ ■■ Do not exceed the recommended DNA load (66 ng/well undigested DNA or 1,500 ng/well digested DNA) Use only approved plates (Eppendorf twin.tec semi-skirted 96-well plates, catalog #951020362) with approved pierceable foil heat seals (Bio-Rad catalog #181-4040) Properly seal the 96-well plate. Under- or over-sealed plates result in oil evaporation during thermal cycling and compromise droplet data quality.
Droplet Digital™ PCR Tips, Assay Considerations, and Troubleshooting Channel 1 concentration, copies/µl 2,000 1,600 1,360 1,260 1,200 1,120 1,120 800 400 0 Replicate 1 Replicate 2 Replicate 3 Staphylococcus aureus Replicate 4 Fig. 9.6. Quadruplicate replicates drawn from the same poorly mixed reaction solution demonstrate inconsistent concentration readings.
Droplet Digital™ PCR Tips, Assay Considerations, and Troubleshooting The temperature-sensitive assay used to generate Figure 9.7 is relatively long and very GC-rich (244 bp, 74% GC).
Droplet Digital™ PCR Tips, Assay Considerations, and Troubleshooting Additional Tips No Concentration Calls on Some Wells If a concentration estimate fails to appear in the concentration chart in QuantaSoft, this indicates the software could not auto-analyze or assign droplets to positive or negative populations using its auto-analysis algorithm, or the well had an unusually low droplet count (<10,000).
Droplet Digital™ PCR Tips, Assay Considerations, and Troubleshooting High-Fluorescence Amplitude Droplets Droplet coalescence can create droplets that are much higher in fluorescence amplitude than the other positive droplets. This can be caused by poor droplet transfer technique or extended storage of the droplets pre– or post–thermal cycling. Adjust the scale on the 1-D or 2-D amplitude charts in order to set the thresholds in these cases.
Droplet Digital™ PCR Tips, Assay Considerations, and Troubleshooting 1 µM A 300 nM 25 nM 100 nM increments 40,000 25 nM increments A07 B07 C08 D07 E07 F07 G07 H07 A09 B09 C09 D09 E09 F09 G09 H09 A11 B11 C11 35,000 Channel 1 amplitude 30,000 25,000 20,000 15,000 10,000 5,000 0 0 50,000 100,000 150,000 200,000 250,000 Event number 300,000 B 22.5 Separation value, arbitrary units 20 17.5 15 12.5 10 7.5 5 2.
Droplet Digital™ PCR Tips, Assay Considerations, and Troubleshooting Wild-type DNA – B07 + B08 C03 – C04 D03 + D05 E03 – E04 F05 + F08 Mutant DNA G06 G07 Channel 1 amplitude 25,000 20,000 15,000 10,000 5,000 0 50,000 100,000 Event number 150,000 200,000 Fig. 9.10. Mutant DNA spiked into increasing amounts of wild-type DNA. As the total added DNA concentration increases, the positive fluorescence amplitudes decrease and the negative fluorescence amplitudes increase.
Appendix A Ordering Information QX200™ Droplet Digital™ PCR (ddPCR™) System 186-4001 QX200 Droplet Digital PCR System, includes droplet generator, droplet reader, laptop computer, software, associated component consumables 186-4002 QX200 Droplet Generator, includes droplet generator, 1 pkg of 24 DG8™ cartridges, 1 pkg of 24 DG8 gaskets, 2 cartridge holders, 1 power cord 186-4003 QX200 Droplet Reader, includes droplet reader, ddPCR manual, 2 plate holders, USB cable, power cord 186-4007 Droplet Ge
Appendix A: Ordering Information 186-4005 Droplet Generation Oil for EvaGreen, 2 x 7 ml 186-4006 Droplet Generation Oil for EvaGreen, 10 x 7 ml 186-3004 ddPCR Droplet Reader Oil, 2 x 1 L ddPCR Reagents 186-3026 dPCR Supermix for Probes, 2 ml (2 x 1 ml), 200 x 20 µl reactions, d 2x supermix 186-3010 d dPCR Supermix for Probes, 5 ml (5 x 1 ml), 500 x 20 µl reactions, 2x supermix 186-3027 ddPCR Supermix for Probes, 25 ml (5 x 5 ml), 2,500 x 20 µl reactions, 2x supermix 186-3028 ddPCR Super
Appendix A: Ordering Information 186-4052 QX200 Buffer Control Kit for EvaGreen, 9 ml (2 x 4.
Appendix B Technical Error Bars in Droplet Digital™ PCR Because Droplet Digital PCR (ddPCR™) is a digital counting system, you can calculate the measurement error based on the droplet data from a single well. This is the technical error (also referred to as the Poisson error in QuantaSoft™ software), and it is a good estimate of the errors you can expect to see on true technical replicates (defined as aliquots of the same starting material loaded into multiple ddPCR wells).
Appendix B: Technical Error Bars in Droplet Digital™ PCR Whenever you subsample from a larger volume with the intent to measure properties of the whole volume, random effects will lead to slightly different measurements from the subsampled volume. Subsampling error is most significant at low concentrations.
Appendix B: Technical Error Bars in Droplet Digital™ PCR Partitioning The second contribution to the technical error bars comes from partitioning of the DNA targets into droplets. This is the error that dominates at high concentration. The illustration in Figure 2 shows 288 target molecules partitioned into 144 droplets. These grids are snapshots in time: imagine molecules bouncing around in the sample, then at any given instant divide the sample into 288 partitions.
Appendix B: Technical Error Bars in Droplet Digital™ PCR Figure 4 illustrates the technical error as a function of CPD for 10,000 droplets. Note that the coefficient of variation (CV) is extremely low across the entire range covered here. The dotted lines show the region with CV <2.5%: 0.17–5.1 CPD. The black and blue curves match very closely at low CPD, meaning that subsampling error is more significant than the partitioning error in this example.
Appendix C Acronyms Acronyms ABS — absolute quantification BLAST — Basic Local Alignment Search Tool bp — base pair cDNA — complementary DNA CN — copy number CNV — copy number variation CPD — copies per droplet CV — coefficient of variation ddPCR™ — Droplet Digital™ PCR DNA — deoxyribonucleic acid dsDNA — double-stranded DNA dUTP — 2'-deoxyuridine 5'-triphosphate ERBB2 — human epidermal growth factor receptor 2 gene (also, HER2) FFPE —formalin-fixed, paraffin-embedded FISH — fluorescence in situ hybridiza
Appendix C: Acronyms GMO — genetically modified organism HER2 — human epidermal growth factor receptor 2 gene (also, ERBB2) HIV — human immunodeficiency virus IHC — immunohistochemistry LNA — locked nucleic acid LoD — limit of detection LoQ — limit of quantification miRNA — microRNA NGS — next-generation sequencing NTC — no template control PBMC — peripheral blood mononuclear cell PCR — polymerase chain reaction qPCR — quantitative PCR RED — rare target sequence detection (rare event detection) RFU — relat
Index B A ABS 19, 20, 28, 29, 59, 98 base composition 11 absolute quantification 1, 2, 7, 8, 17, 19, 20, 28, 29, 54, 59, 68, 74 Basic Local Alignment Search Tool 15 adverse drug response 37 biomarker 47 algorithm 6, 29, 87 biopsy 39, 46, 48, 53, 94 breast cancer 39 Poisson 6–10, 29, 33, 35, 36 BLAST 15, 98 Ambion 60, 62 Primer-BLAST 80 human brain reference 60 amplicon 13, 15–17, 24, 25, 27, 28, 40, 42, 46, 51–53 blood draw 56 reference 28, 40, 42 amplification 2, 4, 7, 11, 12
Index design 11–13, 15, 19, 40, 46, 51, 53, 59, 66, 73, 78, 80, 86 channel 5, 6, 9, 19, 23–27, 48–51, 59, 66, 67, 69, 74–76, 78, 79–83, 85, 87, 89, 90 assay 11, 13, 15, 40, 46, 53, 73, 78, 86 chloroform 10 experimental 19, 51, 53, 59 Chr10q1 76, 77 primer 11, 12, 80 Chr13q3 76, 77 detection of rare sequences 7 chromosome 73, 74, 76 detection system 4, 56 chromosome 6 74 chromosome 10 73, 74, 76 DG8 17, 18, 31, 52, 81, 83, 84, 91 clonally derived Down syndrome digital PCR 1
Index footprint 16 drug response 37 amplicon 16 adverse 37 dUTP 69, 81, 92, 93, 98 formalin-fixed, paraffin-embedded 17, 40, 53, 58, 98 dyes 3, 11–13, 24, 59 formula 7, 14, 16, 35 dsDNA 12, 24, 98 assay 59 intercalating 3 fragmentation 16, 17, 40 E salt correction 14 G EEF2 61–63 GAPDH 62, 63 electrophoresis 64, 70 GAPDH-PL 62 endonuclease 16, 43 GC 11–14, 86, 98 enhancers 13, 46 gDNA 9, 10, 16, 43, 46, 87, 98 Tm 13 gene expression 7, 24, 26, 46, 57, 59–63 enzyme 16, 28, 42
Index human epidermal growth factor receptor 2 gene 60, 98, 99 microarrays 37 human immunodeficiency virus 46, 99 microfluidic 83 SNP-based 37 microfluidics 2, 3 hybridization 37, 60, 63, 98 mir-210 74, 75 comparative genomic 37 miRNA 74, 75, 99 hydrolysis probes 3, 12, 13, 58 synthetic template 74 I MIT 13, 15 IHC 60, 62, 63, 99 mosaicism 39 intercalating dyes 3 somatic 39 in vitro transcribed RNA 60, 99 MRGPRx1 38, 72, 73 iScript™ 58, 60 multiplexing 24, 27, 75, 76 advanced cDNA syn
Index QuantaSoft software 10, 19, 20, 22, 29–31, 35, 36, 39, 41, 59, 67, 69, 77, 79, 87 partitioning 2, 3, 7, 9, 28, 30, 33, 38, 41, 47, 53, 58, 64, 94, 96, 97 error 96, 97 quantitative PCR 11, 36, 37, 51, 57, 99 PBMC 55, 56, 99 PCR 2–4, 6–8, 11, 13, 15–20, 23, 24, 27, 30, 31, 40, 47, 52, 54, 56–58, 64–69, 74, 78, 80, 81, 82, 84, 86, 88, 90, 93, 99 quencher 12, 13 QX100 1–5, 12, 13, 16–20, 28, 53, 55, 66, 68, 69, 71, 75, 83, 91, 93 QX200 1–4, 11–13, 17–20, 24, 26, 28, 53, 66, 69, 75, 83, 91–93 ampli
Index structure 11, 12, 15, 42, 57, 59, 60, 83, 87 RT-ddPCR 3, 58–60, 82, 92, 99 RT-qPCR 57, 58, 60, 99 one-step 57 two-step 57, 60 subsampling error 95–97 S supermix 3, 17, 18, 24, 26, 28, 31, 58, 69, 75, 77, 81–83, 92, 93 salt 12, 14, 16, 42 concentration 12, 16, 42 sample 2, 3, 5–8, 10–13, 15–20, 22–24, 26, 29–32, 36, 38–41, 44, 46, 47, 49, 51–58, 60–64, 67, 71–74, 78–96 secondary 11, 12, 15, 57, 59, 60, 83, 87 supernatant 83 T TaqMan 3, 11–13, 17, 46, 58, 62, 66, 74, 75, 83 heterogeneo
Index U UDG 81, 99 ultraconserved sequences 40 ultramers 82 UNG 81, 82, 99 uracil DNA glycosylase 81, 99 uracil N-glycosylase 81, 99 uracils 82 V VIC 3, 4, 6, 9, 10, 13, 20, 22, 40, 46, 48, 51, 59, 61, 73, 74, 78, 82 virus 46, 55, 56 W Well Editor 19, 29, 59 Whitehead Institute for Biomedical Research 13 wild-type 7, 40, 46–53, 88, 90 clusters 46 sequences 47, 48 106 | Droplet Digital™ PCR Applications Guide
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