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Droplet Digital PCRDroplet Digital™ PCR Applications Guide

6 | Droplet Digital™ PCR Applications GuideDroplet Digital™ PCRFig. 1.10. 2-D plot of droplet fluorescence.Droplet Digital PCR data from a duplex exper

96 | Droplet Digital™ PCR Applications GuideAppendix B: Technical Error Bars in Droplet Digital™ PCRPartitioningThe second contribution to the technic

Droplet Digital™ PCR Applications Guide | 97 Fig. 4. Relative contribution of partitioning and subsampling errors to ddPCR error. CPD, copies per dr

98 | Droplet Digital™ PCR Applications Guide Appendix C AcronymsAcronymsABS — absolute quantificationBLAST — Basic Local Alignment Search Toolbp —

Droplet Digital™ PCR Applications Guide | 99Appendix C: AcronymsGMO — genetically modified organismHER2 — human epidermal growth factor receptor 2 ge

100 | Droplet Digital™ PCR Applications Guide IndexAABS 19, 20, 28, 29, 59, 98absolute quantification 1, 2, 7, 8, 17, 19, 20, 28, 29, 54, 59, 68, 7

Droplet Digital™ PCR Applications Guide | 101channel 5, 6, 9, 19, 23–27, 48–51, 59, 66, 67, 69, 74–76, 78, 79–83, 85, 87, 89, 90 color 67, 75chlo

102 | Droplet Digital™ PCR Applications GuideIndexdrug response 37 adverse 37dsDNA 12, 24, 98dUTP 69, 81, 92, 93, 98dyes 3, 11–13, 24, 59 assay

Droplet Digital™ PCR Applications Guide | 103human epidermal growth factor receptor 2 gene 60, 98, 99human immunodeficiency virus 46, 99hybridizat

104 | Droplet Digital™ PCR Applications GuideIndexpartitioning 2, 3, 7, 9, 28, 30, 33, 38, 41, 47, 53, 58, 64, 94, 96, 97 error 96, 97PBMC 55, 56,

IndexDroplet Digital™ PCR Applications Guide | 105 RT-ddPCR 3, 58–60, 82, 92, 99RT-qPCR 57, 58, 60, 99 one-step 57 two-step 57, 60Ssalt 12, 1

Droplet Digital™ PCR Applications Guide | 7Emerging Applications of Droplet Digital PCRSample partitioning allows the sensitive, specific detection o

Index106 | Droplet Digital™ PCR Applications GuideUUDG 81, 99ultraconserved sequences 40ultramers 82UNG 81, 82, 99uracil DNA glycosylase 81, 99u

For more information, visit www.bio-rad.com/web/ddPCRAppGuide.Bio-Rad Technical SupportFor help and technical advice, please contact the Bio-Rad Tech

8 | Droplet Digital™ PCR Applications GuideDroplet Digital™ PCRFig. 1.12. Estimating target concentration by ddPCR.ddPCR is an end-point measurement t

Droplet Digital™ PCRDroplet Digital™ PCR Applications Guide | 9Figure 1.13 shows the effect of varying the threshold value (Figure 1.13A) on concent

10 | Droplet Digital™ PCR Applications GuideDroplet Digital™ PCRFig 1.14. QuantaSoft software concentration estimates. Twofold (A) and 10% (B) dilutio

Droplet Digital™ PCR Applications Guide | 112 Designing Droplet Digital™ PCR ExperimentsAssay Design for Droplet Digital PCRAs with any PCR-based

12 | Droplet Digital™ PCR Applications GuideWhen designing primers for a target sequence, follow these guidelines: Design primers that have a GC cont

Droplet Digital™ PCR Applications Guide | 13Designing Droplet Digital™ PCR ExperimentsFig. 2.1. In TaqMan assays, hydrolysis probes are hydrolyzed b

14 | Droplet Digital™ PCR Applications GuideFig. 2.2. Main tab in Primer3Plus.In the Main window, paste your target DNA sequence in the “Paste source

Droplet Digital™ PCR Applications Guide | 15Designing Droplet Digital™ PCR ExperimentsFig. 2.3. Assay design output.Several important design feature

iii | Droplet Digital™ PCR Applications GuideTable of ContentsChapter 1 Droplet Digital™ PCR ...

16 | Droplet Digital™ PCR Applications GuideFor best results, restriction digestion of your DNA sample outside of the amplicon region is recommended.

Droplet Digital™ PCR Applications Guide | 17Designing Droplet Digital™ PCR ExperimentsThe example is relevant to any gene that is present at the nor

18 | Droplet Digital™ PCR Applications GuideFig. 2.4. Loaded DG8 cartridge.Fig. 2.5. Loaded DG8 cartridge placed in the QX200 droplet generator.Note:

Droplet Digital™ PCR Applications Guide | 19Fig. 2.6. Labeling the plate.After heat sealing, place the PCR plate in a thermal cycler for PCR using t

20 | Droplet Digital™ PCR Applications GuideDroplet ReadingFollowing PCR amplification of the nucleic acid target in the droplets, place the PCR plate

Droplet Digital™ PCR Applications Guide | 21Fig. 2.7. Setting thresholds in a 1-D plot.Note: Use 1-D plots for temperature gradient experiments and

22 | Droplet Digital™ PCR Applications GuideFig. 2.8. Setting thresholds in a 2-D plot.Fig. 2.9. Merged well data.For correct quantification of a duple

Droplet Digital™ PCR Applications Guide | 23Designing Droplet Digital™ PCR ExperimentsFig. 2.10. Thermal gradient optimization.PCR Optimization Usin

24 | Droplet Digital™ PCR Applications GuideDesigning Droplet Digital™ PCR ExperimentsddPCR Using the QX200 System and EvaGreen dsDNA DyeThe QX200 sys

Droplet Digital™ PCR Applications Guide | 25Designing Droplet Digital™ PCR ExperimentsIn Figure 2.13, a gradient of annealing/extension temperatures

Table of ContentsDroplet Digital™ PCR Applications Guide | iv Statistics of ddPCR ...30Co

26 | Droplet Digital™ PCR Applications GuideDesigning Droplet Digital™ PCR ExperimentsFig. 2.14. Splice variant discrimination using the QX200 ddPCR E

Droplet Digital™ PCR Applications Guide | 27Multiplexing with EvaGreenDifferences in droplet amplitude due to differences in amplicon length or opti

28 | Droplet Digital™ PCR Applications Guide3 Absolute Quantification and the Statistics of Droplet Digital™ PCRRunning Absolute Quantification Exper

Droplet Digital™ PCR Applications Guide | 29Fig. 3.1. Example of a well-performing assay for absolute quantification.Absolute Quantification and the S

Absolute Quantification and the Statistics of Droplet Digital™ PCR30 | Droplet Digital™ PCR Applications GuideFig. 3.2. Sample concentrations are plo

Droplet Digital™ PCR Applications Guide | 31Copies per MicroliterQuantaSoft software provides concentration results in copies of target per microlit

Absolute Quantification and the Statistics of Droplet Digital™ PCR32 | Droplet Digital™ PCR Applications GuideFig. 3.3. Outcome of ddPCR in two modes

Droplet Digital™ PCR Applications Guide | 33High Concentration ExampleConsider the case in which 50,000 target molecules are present in 20 µl (50,00

Absolute Quantification and the Statistics of Droplet Digital™ PCR34 | Droplet Digital™ PCR Applications GuideEven at an average of 5 copies/droplet (t

Droplet Digital™ PCR Applications Guide | 35 Absolute Quantification and the Statistics of Droplet Digital™ PCRFormula for Calculating ConcentrationT

Table of Contentsv | Droplet Digital™ PCR Applications GuideOne-Step RT-ddPCR Kit for Probes ...58Dat

Absolute Quantification and the Statistics of Droplet Digital™ PCR36 | Droplet Digital™ PCR Applications GuideErrors in ddPCRTwo types of errors are re

Droplet Digital™ PCR Applications Guide | 374 Copy Number Variation AnalysisOverviewAnalysis of copy number (CN) involves determining the number o

38 | Droplet Digital™ PCR Applications GuideFig. 4.1. Discrimination between consecutive CN states is more difficult at higher order copy numbers.Fig.

Droplet Digital™ PCR Applications Guide | 39Copy Number Variation AnalysisCNV CalculationsCNV analysis by ddPCR involves quantification of target and

40 | Droplet Digital™ PCR Applications GuideTable 4.1. Detection of CN variant cells in a heterogeneous sample. Sensitivity Required for CN Determinat

Droplet Digital™ PCR Applications Guide | 41Copy Number Variation AnalysisRunning a CNV AssaySelect Copy Number Variation (CNV) as the experiment ty

42 | Droplet Digital™ PCR Applications GuideConsiderations in planning a restriction digestion. Do not cut the target or reference amplicon Choose a

Droplet Digital™ PCR Applications Guide | 43Copy Number Variation AnalysisLists of recommended restriction enzymes for CNV ddPCR are provided in Tab

44 | Droplet Digital™ PCR Applications GuideCopy Number Variation AnalysisTable 4.5. Sample loading considerations for higher-order CN targets (10–50

Droplet Digital™ PCR Applications Guide | 455 Rare Mutation and Sequence DetectionOverviewDroplet Digital™ PCR (ddPCR™) enables detection and analy

Droplet Digital™ PCR Applications Guide | 11 Droplet Digital™ PCRIntroductionDroplet Digital polymerase chain reaction (ddPCR™) was developed to p

46 | Droplet Digital™ PCR Applications GuideFig. 5.1. Rare mutation detection and rare sequence detection assays. Rare Mutation and Sequence Detection

Droplet Digital™ PCR Applications Guide | 47Rare Mutation and Sequence DetectionFigure 5.2 depicts a bulk 20 μl solution containing 40 mutants and 4

48 | Droplet Digital™ PCR Applications GuideRare Mutation and Sequence DetectionRMD Experiment ConsiderationsThe first consideration for low-level dete

Droplet Digital™ PCR Applications Guide | 49Rare Mutation and Sequence DetectionOnce the best melting temperatures have been identified using 1-D plo

50 | Droplet Digital™ PCR Applications GuideRare Mutation and Sequence DetectionFig. 5.5. 2-D amplitude plot of a wild-type control well containing 5,

Droplet Digital™ PCR Applications Guide | 51Rare Mutation and Sequence DetectionFig. 5.7. 2-D plot of BRAF V600E assay. 0 1,000 2,000 3,000 4,00

52 | Droplet Digital™ PCR Applications GuideRare Mutation and Sequence DetectionAdditional wells may be screened to ensure detection of more than one

Droplet Digital™ PCR Applications Guide | 53Rare Mutation and Sequence DetectionWe recommend always running negative controls to monitor for all pos

54 | Droplet Digital™ PCR Applications GuideRare Mutation and Sequence DetectionRSD Experimental StrategiesSome RSD applications require reliable quan

Droplet Digital™ PCR Applications Guide | 55Rare Mutation and Sequence DetectionFig. 5.9. Detection of a very low target concentration in a high bac

2 | Droplet Digital™ PCR Applications GuideddPCR has the following benefits for nucleic acid quantification: Unparalleled precision — the massive sampl

56 | Droplet Digital™ PCR Applications GuideIn the example given, the measurement error (CV) is introduced at the time of subsampling (at the time of

Droplet Digital™ PCR Applications Guide | 576 Gene ExpressionOverviewReverse transcription quantitative PCR (RT-qPCR) is a commonly used method in

58 | Droplet Digital™ PCR Applications GuideGene ExpressionSimilar to RT-qPCR, reverse transcription Droplet Digital™ PCR (RT-ddPCR) approaches can al

Droplet Digital™ PCR Applications Guide | 59Gene ExpressionThe one-step RT-ddPCR kit for probes is formulated for efficient and sensitive reverse tra

60 | Droplet Digital™ PCR Applications GuideGene ExpressionFig. 6.2. Comparison of one-step RT-ddPCR and two-step RT-ddPCR with in vitro transcribed

Droplet Digital™ PCR Applications Guide | 61Gene ExpressionFigure 6.3 illustrates the determination of ERBB2 transcript levels relative to the EEF2

62 | Droplet Digital™ PCR Applications GuideGene ExpressionFigure 6.5 is an example of normal breast tissue compared with HER2+ tissues (fresh frozen

Droplet Digital™ PCR Applications Guide | 63Gene ExpressionTable 6.1 shows a comparison of IHC, FISH, genomic copy number, and transcript level in c

64 | Droplet Digital™ PCR Applications Guide7 Next-Generation Sequencing Library AnalysisOverviewNext-generation sequencing (NGS) systems are extremel

Droplet Digital™ PCR Applications Guide | 65 Next-Generation Sequencing Library AnalysisddPCR Quantification on Illumina TruSeq v2 ChemistryFor the I

Droplet Digital™ PCR Applications Guide | 3Fig. 1.4. The droplets created by the QX200 droplet generator are uniform in size and volume.Droplet Gene

Next-Generation Sequencing Library Analysis66 | Droplet Digital™ PCR Applications GuideFig. 7.2. Design of the ddPCR library quantification kit for Ill

Droplet Digital™ PCR Applications Guide | 67Next-Generation Sequencing Library AnalysisDroplets that appear above and below the insert population (l

Next-Generation Sequencing Library Analysis68 | Droplet Digital™ PCR Applications GuideFig. 7.6. Plot of the reads vs. input library concentration. Im

Droplet Digital™ PCR Applications Guide | 69Next-Generation Sequencing Library AnalysisFig. 7.7. ddPCR library balancing results. PF, passing filter.

Next-Generation Sequencing Library Analysis70 | Droplet Digital™ PCR Applications Guide3. Add 20 µl of TE buffer for each well used; add additional T

Droplet Digital™ PCR Applications Guide | 71 Next-Generation Sequencing Library Analysis1,500 —850 —700 —500 —400 —300 —200 —150 —100 —50 —15 —bp bp

72 | Droplet Digital™ PCR Applications Guide8 Additional ApplicationsLinkage AnalysisPhysical linkage of two alleles in Droplet Digital™ PCR (ddPCR™)

Droplet Digital™ PCR Applications Guide | 73Additional ApplicationsFig. 8.1. CNV of different human samples using restriction digestion vs. undigest

74 | Droplet Digital™ PCR Applications GuideAdditional ApplicationsDemonstration of a milepost assay is presented in Figure 8.3, which shows the 2-D p

Droplet Digital™ PCR Applications Guide | 75Additional ApplicationsFig. 8.4. Day-to-day reproducibility of mir-210 miRNA assay: measured vs. theoret

4 | Droplet Digital™ PCR Applications GuideDroplet Digital™ PCRFig. 1.5. The C1000 Touch thermal cycler provides robust performance for ddPCR experime

Additional Applications76 | Droplet Digital™ PCR Applications GuideFig. 8.5. Dilution series of an assay targeting chromosome 10 of the human genome.C

Droplet Digital™ PCR Applications Guide | 77 Additional ApplicationsIn this experiment, a single FAM assay was used (RPP30) at standard 1x final conc

78 | Droplet Digital™ PCR Applications Guide9 Droplet Digital™ PCR Tips, Assay Considerations, and TroubleshootingAssay-Dependent Cluster ShiftsA

Droplet Digital™ PCR Applications Guide | 79Droplet Digital™ PCR Tips, Assay Considerations, and TroubleshootingFig. 9.1. Cross-reacting probes in a

Droplet Digital™ PCR Tips, Assay Considerations, and Troubleshooting80 | Droplet Digital™ PCR Applications GuideTo prevent the off-target amplification

Droplet Digital™ PCR Applications Guide | 81Droplet Digital™ PCR Tips, Assay Considerations, and TroubleshootingPositive Droplets in No Template Con

Droplet Digital™ PCR Tips, Assay Considerations, and Troubleshooting82 | Droplet Digital™ PCR Applications GuideNote: The UNG approach can address con

Droplet Digital™ PCR Applications Guide | 83Droplet Digital™ PCR Tips, Assay Considerations, and TroubleshootingNo or Few Positive DropletsIf a new,

Droplet Digital™ PCR Tips, Assay Considerations, and Troubleshooting84 | Droplet Digital™ PCR Applications Guide Do not exceed the recommended DNA lo

Droplet Digital™ PCR Applications Guide | 85Fig. 9.6. Quadruplicate replicates drawn from the same poorly mixed reaction solution demonstrate incons

Droplet Digital™ PCR Applications Guide | 5Droplets are spaced out individually for fluorescence reading by the droplet reader (Figure 1.7). Fluores

Droplet Digital™ PCR Tips, Assay Considerations, and Troubleshooting86 | Droplet Digital™ PCR Applications GuideThe temperature-sensitive assay used t

Droplet Digital™ PCR Applications Guide | 87Additional TipsNo Concentration Calls on Some WellsIf a concentration estimate fails to appear in the co

88 | Droplet Digital™ PCR Applications GuideDroplet Digital™ PCR Tips, Assay Considerations, and TroubleshootingHigh-Fluorescence Amplitude Droplets D

Droplet Digital™ PCR Applications Guide | 89Droplet Digital™ PCR Tips, Assay Considerations, and TroubleshootingFig. 9.9. Good separation of positiv

90 | Droplet Digital™ PCR Applications GuideDroplet Digital™ PCR Tips, Assay Considerations, and TroubleshootingReferenceKwok S and Higuchi R (1989).

Droplet Digital™ PCR Applications Guide | 91 Appendix A Ordering InformationQX200™ Droplet Digital™ PCR (ddPCR™) System 186-4001 QX200 Droplet

92 | Droplet Digital™ PCR Applications GuideAppendix A: Ordering Information 186-4005 Droplet Generation Oil for EvaGreen, 2 x 7 ml 186-4006 Droplet G

Droplet Digital™ PCR Applications Guide | 93 186-4052 QX200 Buffer Control Kit for EvaGreen, 9 ml (2 x 4.5 ml), 2x buffer 186-3040 ddPCR Library Q

94 | Droplet Digital™ PCR Applications Guide Appendix B Technical Error Bars in Droplet Digital™ PCRBecause Droplet Digital PCR (ddPCR™) is a digita

Droplet Digital™ PCR Applications Guide | 95Appendix B: Technical Error Bars in Droplet Digital™ PCRFig. 1. Subsampling error due to analyzing part