User manual

Table of Contents
v | Droplet Digital
™
PCR Applications Guide
One-Step RT-ddPCR Kit for Probes .................................................58
Data Analysis ...............................................................59
ddPCR Gene Expression Data ..................................................60
HER2 Study ...................................................................60
Data Analysis Results ........................................................60
Chapter 7 Next-Generation Sequencing Library Analysis ......................................64
Overview .....................................................................64
ddPCR Quantification on Illumina TruSeq v2 Chemistry ..................................65
Library Quality Analysis ..........................................................66
Next-Generation Sequencing Reads ................................................67
Library Balancing ...............................................................68
Amplicon Recovery from Droplets ..................................................69
Chapter 8 Additional Applications .........................................................72
Linkage Analysis ...............................................................72
Milepost Assay ..............................................................73
microRNA Amplification by ddPCR .................................................74
Day-to-Day Reproducibility Study: mir-210 miRNA ...................................75
Multiplexing ................................................................75
Chapter 9 Droplet Digital
™
PCR Tips, Assay Considerations, and Troubleshooting ..................78
Assay-Dependent Cluster Shifts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .78
Shifted Clusters Due to Probe Cross-Reactivity .....................................78
Probe Cross-Reactivity Can Identify Off-Target Amplification ..............................79
Positive Droplets in No Template Control Wells ........................................81
High Mean Fluorescence Amplitude Intensity ..........................................82
No or Few Positive Droplets .......................................................83
No or Low Total Droplet Count ....................................................83
Inconsistent Concentration Results .................................................84
Insufficient Mixing ............................................................84
Effects of Poor Cycler Uniformity ................................................85
Concentrations Consistently Lower than Predicted .....................................86
Additional Tips .................................................................87
No Concentration Calls on Some Wells ...........................................87
Target Accessibility ...........................................................87
High-Fluorescence Amplitude Droplets ............................................88
Troubleshooting EvaGreen ddPCR Reactions .........................................88
Reference ....................................................................90
Appendix A Ordering Information ..........................................................91
QX200
™
Droplet Digital
™
PCR (ddPCR
™
) System ......................................91
ddPCR Reagents ..............................................................92
Thermal Cycler and Plate Sealer ...................................................93
Appendix B Technical Error Bars in Droplet Digital
™
PCR .......................................94
Subsampling ..................................................................94
Partitioning. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .96
Appendix C Acronyms ...................................................................98
Index ...........................................................................100