ddpcrquant Hands-on Wim Trypsteen Matthijs Vynck Doctoral schools specialist course 1 september 2016, t Pand Ghent
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1 ddpcrquant Hands-on Wim Trypsteen Matthijs Vynck Doctoral schools specialist course 1 september 2016, t Pand Ghent 1
2 Day 1 ( ) Day 2 ( ) 08:00-9:30 Introduction to digital PCR Ward De Spiegelaere 09:30-10:00 coffee break coffee break 10:00-12:00 Absolute quantification by dpcr: theory + applications (HIV, excision circles, splice variants) Linos Vandekerckhove Ide Smets 12:00 Lunch Lunch 13:00-14:30 Statistical considerations of dpcr and droplet calling methods Lieven Clement Olivier Thas 14:30-15:00 coffee break coffee break 15:00-17:00 excercises on Shiny app Wim Trypsteen en Matthijs Vynck 17:00-17:15 short break Short break 17:15-18:00 Power analysis for dpcr Olivier Thas Assay Design + Hands on training Jo Vandesompele Multitarget applications: CNV, human and mouse examples, genotyping, mutation detection Jo Vandesompele Nicole Hersmus Patrick Pauwels RNA quantification by dpcr and normalization tools Ward De Spiegelaere Nicole Hersmus GLMM models for dpcr + Hands on Lieven Clement Olivier Thas Tips and Tricks Minimal reporting guidelines Ward De Spiegelaere 2
3 PMID: ddpcrquant (Global) Goals GOALS 1. Introduce everyone to the ddpcrquant tool 2. Independent analysis of ddpcr data 3 Trypsteen et al. Anal Bioanal Chem Jul;407(19): PMID:
4 Outline ddpcrquant overview Annotation Requirements Demo + DIY Analysis Exporting Data from the ddpcr Reader Running ddpcrquant Exercises part 1 and 2 Head file options 4
5 ddpcrquant HEAD file Summary of the experiment Contains annotation info (NTC,..) Well ExptType Experiment Sample TypeAssay Assay Status A01 Absolute Quantification Absolute Quantitation template dil 160 Ch1Unknown RU5 OK A02 Absolute Quantification Absolute Quantitation template dil 10 Ch1Unknown RU5 OK A03 Absolute Quantification Absolute Quantitation template dil 160 Ch1Unknown LTR GAG PETRA OK AMPLITUDE files Individual well files with the fluorescent intensity information (droplets) Assay1 Amplitude Assay2 Amplitude Cluster
6 6 ddpcrquant
7 ddpcrquant Fisher-Tippett theorem: The distribution of block maxima is given by the Generalized Extreme Value distribution (GEV) = Block maxima (extremes) follow this family of distributions 7
8 ddpcrquant D: Template/Mix Well assay name type positive droplets negative droplets total droplets concentration lowerci upperci 1 merged 2 LTR merged_ntc ntc E01 2 LTR gdna 1 sample F01 2 LTR gdna2 sample G01 2 LTR plas 1 sample H01 2 LTR plas 2 sample
9 Limitations ddpcrquant is made for 1-D analysis (Bio-Rad qx100/qx200) 1 channel at a time If 2 channels per well => one after the other will be analyzed Use the NTCs to calculate a single threshold Whether the extreme value measured in the NTC is a real or false positive value is beyond the scope of ddpcrquant ddpcrquant uses a predefined annotation Throws most of the errors when using the tool R functions available but webtool GUI 9
10 Outline ddpcrquant overview Annotation Requirements Demo + DIY Analysis Exporting Data from the ddpcr Reader Running ddpcrquant Exercises part 1 and 2 Head file options 10
11 Annotation Requirements Annotation before run starts: Quantasoft Annotation after run Change headfile in text format reader notepad, noteblock, NOT excel Use qx100/qx200 template 11
12 Annotation Requirements Before the ddpcr run: Annotation & Input Info SampleName: NTC/ntc can only be in the name of the negative control AssayName: different assay names required Type: Select Ch1Unknown or Ch2Unknown (nothing else at the moment) WellNumber: automatically 12 This info will be stored in the Head file
13 Outline ddpcrquant overview Annotation Requirements Demo + DIY Analysis Exporting Data from the ddpcr Reader Running ddpcrquant Exercises part 1 and 2 Head file options 13
14 Exporting Data from Quantasoft After ddpcr run: Load ddpcr data into Quantasoft head QS Raw data 14
15 15 Exporting Data from Quantasoft
16 16 Exporting Data from Quantasoft
17 Exporting Data from Quantasoft After exporting the folder should look like: head QS Amplitude csv 17
18 Exporting Data from Quantasoft This is how the csv file should look like On the first row: 3 names seperated by 2 commas Next rows: 3 numerical values separated by 2 commas Depending on the measured channels 1 or 2 decimal numbers Cluster number 18
19 Exporting data from Quantasoft Decimal sign needs to be. Control Panel > Regional and Language options > Format tab 19
20 Outline ddpcrquant overview Annotation Requirements Demo + DIY Analysis Exporting Data from the ddpcr Reader Running ddpcrquant Exercises part 1 and 2 Head file options 20
21 Running ddpcrquant Where to find the tool and additional info: 1. Browse to Click on the ddpcrquant webtool link (demo) 2. See also the Quick ddpcrquant analysis guide What you will need today: 1. There is a preloaded demo dataset 2. Download the exercises folder from the course dropbox 21
22 Outline ddpcrquant overview Annotation Requirements Demo + DIY Analysis Exporting Data from the ddpcr Reader Running ddpcrquant Exercises part 1 and 2 Head file options 22
23 Exercises part 1 1. Perform the demo analysis (set to qx100!) 2. Use the demo dataset and set the threshold to a manual defined threshold for assay RNA 1. Download the plots, summary table and summary parameters 23
24 Exercises part 2 1. Quantasoft analysis in following folder exercises_part2 1. Open in Quantasoft (qlp) 2. Go to Analyze (left side panel) 1. Select all samples (*) + sort on target + select all RU5 + update 2. Go to 1-D Amplitude 3. Auto Analyze with individual and combined wells => What is wrong? 1. Individual wells? 2. Combined wells? 4. Select wells C01 and E03 and look in ch1 => What is wrong? 5. Write down the concentration of C01 3. Export the amplitudefiles 1. Go to Setup > options > export amplitude and cluster data 2. Select folder 24
25 Exercises part 2 2. Upload the amplitudefiles + headfile to ddpcrquant 3. Run analysis with standard settings Write down the concentration of sample C01 (0.5 vs 0.28) => reason? 4. Run analysis with a threshold What happens with the threshold? Why? 25
26 Outline ddpcrquant overview Annotation Requirements Demo + DIY Analysis Exporting Data from the ddpcr Reader Running ddpcrquant Exercises part 1 and 2 Head file options 26
27 Head file options Annotation before run starts: Quantasoft Annotation after run Change headfile in text format reader: notepad, noteblock, NOT excel Use qx100/qx200 template 27
28 Head file options Open following headfile: 1. Find some missing or wrong annotation and change it according to the annotation rules 6 mistakes 2. Upload head & amplitude and look at overview Demo: Make template 1. Make the demo template yourself Wells: A03,B03,G03,H03 Sample: test1,test1,ntc,ntc TypeAssay: Ch1Unknown Assay: RPP30 2. Upload template & amplitudefiles to ddpcrquant make sure the template file is uploaded first (sort folder before upload) 28
29 Hope it works most of the time! Good luck with future analysis! Troubleshooting + ddpcr advice: Wim.trypsteen@ugent.be 29
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